Variability of the molecular mass of α-subunit in phycocyanins of colonial Microcystis aeruginosa f. aeruginosa in different eutrophic pond and lakes

Electrospray ionization mass spectra of phycocyaninsof eight samples from natural blooms of Microcystis aeruginosa f. aeruginosa collectedat different eutrophic pond and lakes indicated thatthe subunits of phycocyanins in the samespecies have different molecular masses, whereas the subunits have almost constant molecularmasses. A negative linear relationship between themolecular mass of subunit of phycocyanin andthe concentration of chlorophyll of natural andcolonial cyanobacterial samples was found. Aninterannual similarity of the subunitmolecular masses of phycocyanins was observed fromsamples collected at the same sampling site at LakeKasumigaura during 1994 and 1995. A locationalvariability of the subunit molecular massesof phycocyanins was also observed among samplescollected at three eutrophic pond and lakes.     

Antigenic polysaccharides of bacteria. 18. Structure of O-specific polysaccharide chains of Pseudomonas aeruginosa 05 (Lányi) lipopolysaccharide

Polysaccharide chains of P. aeruginosa O5a, b, c, O5a, b, d and O5a, d (Lányi classification) lipopolysaccharides contain D-xylose, N-acetyl-D-fucosamine (FucNAc) and a derivative of 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid, PseN2) carrying acetyl or (R)-3-hydroxybutyryl (Hb) and formyl (Fm) groups as N-acyl substituents. Degradation of the lipopolysaccharides with dilute acetic acid caused depolymerisation of the polysaccharide chains as a result of cleavage of glycosidic linkage of pseudaminic acid to give trisaccharides representing chemical repeating units of the polysaccharides. Basing on analysis of the trisaccharides using 1H and 13C NMR spectroscopy and mass-spectrometry, the following structures of the polysaccharide chains were established: (Formula: see text). O5a, d polysaccharide is identical to P. aeruginosa immunotype 6 O-specific polysaccharide.     

Role of IL-1β in Experimental Cystic Fibrosis upon P. aeruginosa Infection

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1β in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftr ^tm1eur or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1^−/−), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1^−/− double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1β and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1β signaling in response to P. aeruginosa. IL-1β antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1β antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1β production and reduced bacterial clearance. Further, we show that neutralization of IL-1β in d/d mice through the double mutation d/d x IL-1R1^−/− and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1β pathway may be detrimental in CF patients.     

M?ssbauer and EPR spectroscopy of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa has been investigated by EPR and M?ssbauer spectroscopy. Low temperature M?ssbauer data on the native enzyme (Fe3+, S = 5/2) yields a hyperfine field Hsat=-525 kG at the nucleus. This observation is inconsistent with earlier suggestions, based on EPR data of a rubredoxin-like ligand environment around the iron, i.e. a tetrahedral sulfur coordination. Likewise, the dithionite-reduced enzyme has M?ssbauer parameters unlike those of reduced rubredoxin. We conclude that the iron atoms are in a previously unrecognized environment. The ternary complex of the enzyme with 3,4-dihydroxyphenylpropionate and O2 yields EPR signals at g = 6.7 and g = 5.3; these signals result from an excited state Kramers doublet. The kinetics of the disappearance of these signals parallels product formation and the decay of the ternary complex as observed in the optical spectrum. The M?ssbauer and EPR data on the ternary complex establish the iron atoms to be a high-spin ferric state characterized by a large and negative zero-field splitting, D = approximately -2 cm-1.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chains of lipopolysaccharides of P. aeruginosa serogroup O4 (Lányi) and related serotype O6 (Habs) and immunotype 1 (Fisher)

Acidic O-specific polysaccharides were isolated on mild acidic degradation of lipopolysaccharides of Pseudomonas aeruginosa serotypes O4a,b, O4a,c, O4a,d (Lányi classification) and serologically related to them serotype O6 (Habs classification) and immunotype 1 (Fisher classification). The polysaccharides had identical monosaccharide composition and were built up of L-rhamnose, 2-acetamido-2,6-dideoxy-D-glucose,2-formamido-2-deoxy-D-galacturonic acid and 2-acetamido-2-deoxy-D-galactouronamide residues. The latter two derivatives of D-galactosaminuronic acid were found in nature for the first time. All the polysaccharides, but Lányi serotype O4a,c, contained O-acetyl groups. The polysaccharides were readily de-O-acetylated with aqueous triethylamine and de-N-formylated with dilute hydrochloric acid. De-N-formylated polysaccharide of serotype O4a,c was selectively cleaved with nitrous acid upon 2-amino-2-deoxygalacturonic acid residues to form a tetrasaccharide with a 2,5-anhydrotaluronic acid residue on the reducing end. The tetrasaccharide represented a modified repeating unit of the polysaccharide. Solvolysis of all intact polysaccharides with hydrogen fluoride selectively split the glycosidic linkages of 6-deoxy sugars to give the same trisaccharide, including both derivatives of galactosaminuronic acid and having 2-acetamido-2,6-dideoxyglucose on the reducing end. Structural investigation of the oligosaccharides obtained together with methylation analysis and 13C nuclear magnetic resonance data revealed the following structures of the O-specific polysaccharides: (Formula: see text) An independent confirmation of the structures of the repeating units was obtained as the result of full interpretation of the 13C nuclear magnetic resonance spectra of the intact and modified polymers. Spectral data analysis revealed a number of regularities in the effects of glycosidation connecting their values with the anomeric and absolute configuration of pyranose residues. The data on the structures of the O-specific polysaccharides indicated that each of the five P. aeruginosa strains under study should be considered as an individual O-serotype within one O-serogroup.     

Characterization of the χψ subcomplex of Pseudomonas aeruginosa DNA polymerase III

Background

The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth.

Results

Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level.

Conclusions

Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions. When considering splice variants as "natural mutants", evidence on modulation of subcellular localization by variation in domain combination can shed further light on targeting determinants. Although further work will be needed to characterize identified variants, our data might open the route to unravel novel molecular partners and mechanisms, accounting for the multiplicity of functions carried out by the different members of the Longin proteins family.     

Phenogenetic Characterization of a Group of Giant φKZ-like Bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group ofPseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage KZ genome (288 334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1–19]. By DNA homology, the phages were assigned to three species (represented by phages KZ, Lin68, and EL, respectively) and two new genera (KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the KZ species (KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the KZ genus. The origin and evolution of the KZ-like phages are discussed. Analysis of protein sequences encoded by the phage KZ genome made it possible to assume wide migration of the KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

Structure-function relationship of type-IV prepilin peptidase of Pseudomonas aeruginosa – a review

The bifunctional enzyme prepilin peptidase (PilD) from Pseudomonas aeruginosa is a key determinant in both type-IV pilus biogenesis and extracellular protein secretion, in its roles as a leader peptidase and MTase. It is responsible for endopeptidic cleavage of the unique leader peptides that characterize type-IV pilin precursors, as well as proteins with homologous leader sequences that are essential components of the general secretion pathway found in a variety of Gram-negative pathogens. Following removal of the leader peptides, the same enzyme is responsible for the second posttranslational modification that characterizes the type-IV pilins and their homologues, namely N-methylation of the newly exposed N-terminal amino acid residue. This review discusses some of the work begun in order to answer questions regarding the structure-function relationships of the active sites of this unique enzyme.     

The regulation of transport of glucose and methyl α-glucoside in Pseudomonas aeruginosa

The methyl alpha-glucoside-transport system of Pseudomonas aeruginosa has been characterized with respect to its specificity, energy-dependence, kinetics and regulation. The uptake of glucose involved two components, one of which transported glucose (K(m)=8mum) and methyl alpha-glucoside (K(m)=2.8mm) whereas the other was more complex, involving the extracellular activity of glucose dehydrogenase. Mutants defective in this enzyme have been isolated and characterized. The methyl alpha-glucoside-glucose-transport system was repressed when the organism was grown in the absence of glucose; the induction of this transport system by glucose, and its activity once induced, were inhibited by products of citrate metabolism.     

Evidence for M?ssbauer spectroscopy for different forms of iron core in Pseudomonas aeruginosa bacterial ferritin.

Mössbauer spectroscopic studies of whole cells of Pseudomonas aeruginosa, grown under different conditions, indicate that the predominant form of iron in the cells varies significantly. These differences are interpreted in terms of differences in the nature of the iron cores of the bacterial ferritin, which result from different growth conditions.     

Properties of the peptidoglycan-degrading enzyme of the Pseudomonas aeruginosa ϕPMG1 bacteriophage

The ?PMG1 Pseudomonas aeruginosa bacteriophage was isolated. It is characterized by certain peculiarities of the lytic infection cycle and forms a halo (clear zone) around negative colonies. The phage was studied with regard to its potential use in therapeutic phage preparations and as a source of peptidoglycan- and lipopolysacchraide-degrading enzymes. Partial sequencing of the ?PMG1 genome revealed a high degree of homology with the D3 moderate bacteriophage. An open reading frame coding for a lytic transglycosylase has been identified in ?PMG1 genome. The enzyme has been obtained in a recombinant form, and its activity and substrate specificity have been characterized.     

Analyses of Pseudomonas aeruginosa Lectin Binding to α-Galactosylated Glycans

The specificity and binding capacity of the galactophilic lectin from the Gram negative bacterium Pseudomonas aeruginosa (PA-IL) was determined by solid phase measurements using galactosylated neoglycoproteins immobilized on microtiter plates. The bacterial lectin reacted with both short chain (monosaccharide) and long chain (pentasaccharide) glycoconjugates. Among the Galα1-XGal disaccharides, the highest affinity was observed towards the Galα1-3Gal structure. Raising the incubation temperature enhanced the lectin-polysaccharide agglutination, and it is suggested that binding to certain conformations of polysaccharides could vary between lectins with the same monocarbohydrate specificity and that this activity may, in part, be temperature dependent. Histochemical examination of lectin binding to different porcine tissues suggests a differential glycosylation of the carbohydrate antigens on endothelial cells in various parts of the vascular system. In the pancreas, PA-IL also adhered to the excretory ducts. These observations on PA-IL binding could be of importance both to determine infection foci in P. aeruginosa-mediated vacuities and to determine its role for pancreatic involvement in cystic fibrosis.     

Seasonal variations in the vertical distribution and buoyancy of Microcystis aeruginosa Kütz. emend. Elenkin in Rostherne Mere,England

Distribution and buoyancy studies in Rostherne Mere (depth: 30m) show that the life-cycle of Microcystis is intimately related to the cycle of thermal stratification: colonies overwinter on the bottom muds, but migrate to the epilimnion in summer, returning again in late autumn or winter.     

有毒铜绿微囊藻M.aeruginosa pcc7820对氨基酸的吸收

有毒铜绿微囊藻Ｍ．ａｅｒｕｇｉｎｏｓａＰＣＣ７８２０对氨基酸的吸收徐平,曾昭琪（南京大学生物系,２１０００８）ＵＰＴＡＫＥＯＦＡＭＩＮＯＡＣＩＤＳＢＹＴＯＸＩＣＭＩＣＲＯＣＹＳＴＩＳＡＥＲＵＧＩＮＯＳＡＰＣＣ７８２０￥ＸｕＰｉｎｇａｎｄＴｓｅｎｇＣｈ...     

Alteration in virulence characteristics of biofilm cells of Pseudomonas aeruginosa in presence of Tamm-Horsfall protein

Summary Tamm-Horsfall glycoprotein is the most abundant protein in human urine. The present investigation was planned to study the effect of Tamm-Horsfall protein (THP) on elaboration of virulence factors by biofilm cells of Pseudomonas aeruginosa. It was observed that with increase in concentration of THP from 10 to 50 μg/ml there was significant enhancement in elaboration of all the virulence factors by biofilm cells of P. aeruginosa. However, with further increase in concentration of THP from 50 to 70 μg/ml, significant decrease in elaboration of all the virulence traits was observed. Implications of these findings in relation to urinary tract infections caused by P. aeruginosa have been discussed.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of the lipopolysaccharides from P. aeruginosa O1 (Lányi), O3 (Habs), O13 and O14 (Wokatsch), and the serologically related strain NCTC 8505

Lipopolysaccharides from Pseudomonas aeruginosa O1 (Lányi classification), O3 (Habs classification), O13 and O14 (Wokatsch classification), and strain NCTC 8505, which is also related to serogroup O3 (Habs), have structurally similar O-specific polysaccharide chains built up of tetrasaccharide repeating units involving L-rhamnose (Rha), 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-galacturonic acid (GalNAcA), and a di-N-acyl derivative of bacillosamine (BacN): 2,4-diacetamido-2,4,6-trideoxy-D-glucose or 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido]-D-glucose. The latter derivative was obtained free by solvolysis with hydrogen fluoride of carboxyl-reduced Habs O3 polysaccharide, and was identified by 1H-nuclear magnetic resonance spectroscopy and by mass spectrometry of the corresponding methylated alditol. Habs O3, Lányi O1, and Wokatsch O14 polysaccharides contained O-acetyl groups. Solvolysis with hydrogen fluoride of the native Habs O3 polysaccharide resulted in selective cleavage of the glycosidic linkages of 6-deoxy sugars to give the trisaccharide fragment involving all three N-acylated amino sugars. Similar solvolysis of NCTC 8505 polysaccharide afforded a mixture of disaccharide and trisaccharide with N,N'-diacetylbacillosamine at the reducing end. Smith degradation of Habs O3 polysaccharide resulted in selective oxidation of rhamnose to give a glycoside of a trisaccharide with glyceraldehyde as the aglycone. Smith degradation of NCTC 8505 polysaccharide was complicated by the formation of the glycoside of a trisaccharide with an aglycone of unknown structure. A trisaccharide with rhamnose at the reducing end was also isolated after Smith degradation of the latter polysaccharide. Analysis of the composition and structure of all oligosaccharides obtained, and detailed examination of the 13C-nuclear magnetic resonance spectra of these oligosaccharides, and of both intact and modified polysaccharides, revealed the following structures of the repeating units. The structure for the NCTC 8505 polysaccharide differs from that proposed previously [Tahara, Y. and Wilkinson, S.G. (1983) Eur. J. Biochem. 134, 299-304] in the configurations assigned to the glycosidic linkages of rhamnose and bacillosamine. The results obtained show the P. aeruginosa strains studied to represent three different O-serotypes in a single O-serogroup (Formula: see text).     

Chloramphenicol acetyltransferase fromPseudomonas aeruginosa—a new variant of the enzyme

Pseudomonas aeruginosa isolates are highly resistant to chloramphenicol (minimal inhibitory concentration, 100–1,000 g/ml). Most of the strains tested produce the enzyme chloramphenicol acetyltransferase (CAT) while 15% do not produce the enzyme, although they are highly resistant to the drug. In an attempt to understand the resistance mechanism ofP. aeruginosa to chloramphenicol, CAT produced by this microorganism was examined and compared with other known variants of the enzyme which are usually associated with high-level resistance in other species. CAT ofP. aeruginosa was found to be synthesized constitutively and to have a molecular weight of 22,500 per protomer. The specific activity of the enzyme is 30-fold lower than that ofEscherichia coli type II CAT. The same specific activity was obtained for CAT produced by two isolates ofP. aeruginosa, one with high and one with low resistance. The elution patterns from affinity and hydrophobic chromatography,K _m 's for chloramphenicol, the high affinity of the enzyme for acetyl-CoA (2- to 6-fold greater than that of any other variant) and insensitivity to sulfhydryl reagents indicate that the properties of this enzyme are not identical with those of any of the known variants.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

A Study on the Mechanism of DNA Excretion from P. aeruginosa KYU-1—Effect of Mitomycin C on Extracellular DNA Production—

The mechanism of excretion of DNA was investigated using strain KYU-1. This strain produced a considerable amount of DNA extracellularly (7.8mg/ml), and the amount of extracellular DNA per ml of the culture was scores of times more compared to that of intracellular DNA per ml of the culture. DNA synthesis was repressed by the addition of inhibitors (nalidixic acid, 5-fluoro uracil or mitomycin C), their inhibition rates were 35 to 54%. With the addition of 100 μg/ml of mitomycin C at the middle of the logarithmic growth phase, only 0.89 mg of extracellular DNA per ml of the culture was obtained, the inhibition rate was 95%. However, the amount of organic phosphate synthesized in the culture was almost the same no matter whether with or without an inhibitor.

Furthermore, the excretion of DNA from the cell surface of strain KYU-1 was observed with on electron microscope and the DNA molecules excreted were found to be double-stranded.