Studies on the Biosynthesis of α-Putrescinylthymine in Bacteriophage φW-14-Infected Pseudomonas acidovorans

The alpha-putrescinylthymine (putThy) in bacteriophage phiW-14 DNA is synthesized at the mononucleotide level: it is labeled by uracil or deoxyuridine but not by thymidine, and it appears in the acid-soluble pool of infected cells before the onset of phage DNA synthesis. The methylene group at the C-5 position of the pyrimidine moiety of putThy is derived in vivo from a C(1) unit. Extracts of a phage infected thymidine auxotroph of the host, Pseudomonas acidovorans, apparently contain a phage-specific thymidylate synthetase and a phage-specific activity which forms 5-hydroxymethyl dUMP from N(5), N(10)-methylene-tetrahydrofolate and dUMP.     

Cloning and Analysis of the Capsid Morphogenesis Genes of Pseudomonas aeruginosa Bacteriophage D3: Another Example of Protein Chain Mail?

The terminal DNA restriction fragments (PstI-D and -B) of Pseudomonas aeruginosa bacteriophage D3 were ligated, cloned, and sequenced. Of the nine open reading frames in this 8.3-kb fragment, four were identified as encoding large-subunit terminase, portal, ClpP protease, and major head proteins. The portal and capsid proteins showed significant homology with proteins of the lambdoid coliphage HK97. Phage D3 was purified by CsCl equilibrium gradient centrifugation (rho = 1.533 g/ml), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed six proteins with molecular masses of 186, 91, 79, 70, 45, and 32 kDa. The pattern was unusual, since a major band corresponding to the expected head protein (43 kDa) was missing and a significant amount of the protein was retained in the stacking gel. The amino terminus of the 186-kDa protein was sequenced, revealing that the D3 head is composed of cross-linked 31-kDa protein subunits, resulting from the proteolysis of the 43-kDa precursor. This is identical to the situation observed with coliphage HK97.     

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.     

Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chain of the lipopolysaccharide from P. aeruginosa O13 (Lányi)

The O-specific polysaccharide, obtained on mild acid degradation of lipopolysaccharide of Pseudomonas aeruginosa O13 (Lányi classification), is built up of trisaccharide repeating units involving 2-acetamidino-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), 2-acetamidino-2,6-dideoxy-L-galactose (L-fucosacetamidine, L-FucAm), and a new sialic-acid-like sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonuloso n ic acid (Sug), and thus contains simultaneously both acidic and basic functions. Cleavage of the polysaccharide with hydrogen fluoride in methanol revealed the high stability of the glycosidic linkage of the ulosonic acid and afforded methyl glycosides of a disaccharide and a trisaccharide. The structures of the new ulosonic acid and acetamidino group were established by analysing the oligosaccharide fragments by 1H, 13C nuclear magnetic resonance spectrometry, as well as on the basis of their chemical conversions: alkaline hydrolysis of the acetamidino group into acetamido group, reductive deamination with lithium borohydride into the ethylamino group and acetylation with acetic anhydride in pyridine accompanied by intramolecular acylation of the acetamidino function by the ulosonic acid to form a six-membered lactam ring. Identification of the oligosaccharide fragments and comparative analysis of the 13C nuclear magnetic resonance spectra of the oligosaccharides and polysaccharide revealed the following structure of the repeating unit: ----3)D-QuiNAcp(alpha 1----3)Sugp(alpha 2----3)L-FucAmp(alpha 1----.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chains of P. aeruginosa O11 (Lányi) lipopolysaccharides

Lipopolysaccharides were isolated from the phenol layer on aqueous phenol extraction of cells of Pseudomonas aeruginosa O11 (Lányi classification), strains 170021 and 170040. On mild acid degradation of the lipopolysaccharides, with the subsequent gel-filtration on Sephadex G-50, neutral O-specific polysaccharides made up of 6-deoxysugars alone were obtained. Two 2-acetamido-2,6-dideoxy-L-galactose (LFucNAc), 2-acetamido-2,6-dideoxy-D-glucose (DQuiNAc) and L-rhamnose (LRha) residues were found to be the components of the strain 170021 polysaccharide repeating units; those of strain 170040 contained the same monosaccharides, but, instead of 2-acetamido-2,6-dideoxy-D-glucose residue, that of 2-acetamido-2,6-dideoxy-D-galactose (DFucNAc) was present. On the basis of the 13C nuclear magnetic resonance data, methylation analysis and three successive Smith degradations the following structures were determined for the polysaccharide repeating units: strain 170021----2) LRha(alpha 1----3)LFucNAc(alpha 1----3)LFucNAc(alpha 1----3)DQuiNAc(beta 1----; strain 170040,----2)LRha(alpha 1----3)LFucNAc-(alpha 1----3)LFucNAc(alpha 1----3)DFucNAc(beta 1----; differing from one another by configuration of C-4 of 2-acetamido-2,6-dideoxy-D-hexopyranose only.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of the lipopolysaccharides from P. aeruginosa O5 (Lányi) and immunotype 6 (Fisher)

Lipopolysaccharides were isolated from dry bacterial cells of Pseudomonas aeruginosa O5a,b,c, O5a,b,d, O5a,d (Lányi classification) and immunotype 6 (Fisher classification) by the Westphal procedure. Their polysaccharide chains were built up of trisaccharide repeating units containing D-xylose, 2-acetamido-2,6-dideoxy-D-galactose and a new sialic acid-like sugar, the di-N-acyl derivative of 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic (pseudaminic) acid. Formyl, acetyl and (R)-3-hydroxybutyryl groups were identified as the N-acyl substituents of the last monosaccharide; O5a,b,c and O5a,b,d lipopolysaccharides also contained O-acetyl groups. The glycosidic linkage of pseudaminic acid was extremely labile towards acids, and mild acid degradation of the lipopolysaccharides produced, instead of the O-specific polysaccharides, their trisaccharide fragments with pseudaminic acid at the reducing terminus. Similar degradation of immunotype 6 lipopolysaccharides, followed by oxidation with sodium metaperiodate, resulted in a disaccharide fragment due to destruction of xylose. In contrast the glycosidic linkage of pseudaminic acid proved to be more stable towards treatment with hydrogen fluoride than those of xylose and N-acetylfucosamine. As a result, solvolysis of immunotype 6 lipopolysaccharide with hydrogen fluoride in methanol gave methyl glycosides of a disaccharide and a trisaccharide with pseudaminic acid at the non-reducing terminus. Mild acid hydrolysis of these oligosides afforded free 5-N-acetyl-7-N-formylpseudaminic acid, which was identified by the 1H ande 13C nuclear magnetic resonance data, as well as by the mass spectrum of the corresponding fully methylated aldonic acid. As a result of the identification of all oligosaccharides obtained and comparative analysis of the 13C nuclear magnetic resonance spectra of the oligosaccharides and lipopolysaccharides the following structures were established for the repeating units of the polysaccharide chains of the lipopolysaccharides: (Formula: see text) where D-Xyl = D-xylose, D-FucNAc = 2-acetamido-2,6-dideoxy-D-galactose, Pse5N7NFm = 5-amino-3,5,7,9-tetradeoxy-7-formamido-L-glycero-L-manno-nonulosonic+ ++ acid (7-N-formylpseudaminic acid). All the polysaccharides have an identical carbohydrate skeleton and differ from each other by the acyl substituent at N-5 of pseudaminic acid [acetyl or (R)-3-hydroxybutyryl group] or by the presence or absence of the O-acetyl group at position 4 of N-acetylfucosamine. The data obtained account properly for the O specificity of the studied P. aeruginosa strains.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of P. aeruginosa O10 (Lányi) lipopolysaccharides

Mild acid degradation of lipopolysaccharides from Pseudomonas aeruginosa O10a and O10a,b (Lányi classification) resulted in O-specific polysaccharides built up of trisaccharide repeating units containing 2-acetamido-2,6-dideoxy-D-glucose (N-acetylquinovosamine, DQuiNAc), 2-acetamido-2,6-dideoxy-D-galactose (N-acetylfucosamine, DFucNAc), and 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)-3-hydroxybutyramido] -L-glycero-L-manno-nonulosonic acid. The latter is a di-N-acyl derivative of a new sialic-acid-like sugar which was called by us pseudaminic acid (PseN2). A 3-hydroxybutyric acid residue was also found in natural carbohydrates for the first time. In the O10a,b polysaccharide pseudaminic acid carried an O-acetyl group at position 4. For selective cleavage of the O10a polysaccharide, solvolysis with hydrogen fluoride was employed which, owing to the relatively high stability of the glycosidic linkage of pseudaminic acid, led to the disaccharide with this sugar on the non-reducing terminus. Performing the solvolysis in methanol afforded the methyl glycoside of this disaccharide which proved to be more advantageous for further analysis. Carboxyl-reduction made the glycosidic linkage of pseudaminic acid extremely labile, and mild acid hydrolysis of the carboxyl-reduced 010a polysaccharide afforded the trisaccharide with a ketose derivative on the reducing terminus. Establishing the structure of the oligosaccharide fragments obtained and interpreting the 13C nuclear resonance spectra of the polysaccharides allowed to determine the following structure for their repeating units: (formula: see text) In the polysaccharides the N-acetylquinovosamine residue is attached not to pseudaminic acid itself, but to its N-acyl substituent, 3-hydroxybutyryl group, and thus the monomers are linked via both glycosidic and amidic linkages.     

Molecular Characterization of PauR and Its Role in Control of Putrescine and Cadaverine Catabolism through the γ-Glutamylation Pathway in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa PAO1 grows on a variety of polyamines as the sole source of carbon and nitrogen. Catabolism of polyamines is mediated by the γ-glutamylation pathway, which is complicated by the existence of multiple homologous enzymes with redundant specificities toward different polyamines for a more diverse metabolic capacity in this organism. Through a series of markerless gene knockout mutants and complementation tests, specific combinations of pauABCD (polyamine utilization) genes were deciphered for catabolism of different polyamines. Among six pauA genes, expression of pauA1, pauA2, pauA4, and pauA5 was found to be inducible by diamines putrescine (PUT) and cadaverine (CAD) but not by diaminopropane. Activation of these promoters was regulated by the PauR repressor, as evidenced by constitutively active promoters in the pauR mutant. The activities of these promoters were further enhanced by exogenous PUT or CAD in the mutant devoid of all six pauA genes. The recombinant PauR protein with a hexahistidine tag at its N terminus was purified, and specific bindings of PauR to the promoter regions of most pau operons were demonstrated by electromobility shift assays. Potential interactions of PUT and CAD with PauR were also suggested by chemical cross-linkage analysis with glutaraldehyde. In comparison, growth on PUT was more proficient than that on CAD, and this observed growth phenotype was reflected in a strong catabolite repression of pauA promoter activation by CAD but was completely absent as reflected by activation by PUT. In summary, this study clearly establishes the function of PauR in control of pau promoters in response to PUT and CAD for their catabolism through the γ-glutamylation pathway.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of lipopolysaccharides of P. aeruginosa O3 (Lányi), O25 (Wokatsch) and Fisher immunotypes 3 and 7

O-specific polysaccharides, obtained on mild acid degradation of lipopolysacchrides of the serologically related strains Pseudomonas aeruginosa O3 (Lányi classification), O25 (Wokatsch classification) and immunotypes 3 and 7 (Fisher classification), are built up of trisaccharide repeating units involving 2-acetamido-2,6-dideoxy-D-galactose (N-acetyl-D-fucosamine), 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid or 2,3-diacetamido-2,3-dideoxy-L-guluronic acid and 3-acetamidino-2-acetamido-2,3-dideoxy-D-mannuronic acid or 3-acetamidino-2-acetamido-2,3-dideoxy-L-guluronic acid. Lányi O3(a),3d,3f and Wokatsch O25 polysaccharides contain also O-acetyl groups. On the basis of solvolysis with anhydrous hydrogen fluoride, resulting in trisaccharide fragments with N-acetylfucosamine residue at the reducing terminus, chemical modifications of the acetamidino group (alkaline hydrolysis to the acetamido group or reductive deamination to the ethylamino group), as well as analysis by 1H-NMR (including nuclear Overhauser effect experiments) and 13C-NMR spectroscopy, and fast-atom bombardment mass spectrometry, it was concluded that the repeating units of the polysaccharides have the following structures: (Formula: see text) where HexNAcAmA = alpha-L-GulNAcAmA (approximately 70%) or beta-D-ManNacAMA (approximately 30%). Lányi O3(a),3d,3f polysaccharide involves two types of repeating units, which differ from each other only in the configuration at C-5 of the 3-acetamidino-2-acetamido-2,3-dideoxyuronic acid residue. Lányi O3(a),3c,O3a,3d,3e and Fisher immunotypes 3 and 7 polysaccharides contain, together with the major repeating units shown above, a small proportion of units in which the derivative of alpha-L-guluronic acid is replaced by the corresponding beta-D-manno isomer. The data obtained provide the opportunity to substantiate the serological interrelations between these strains of P. aeruginosa by the presence in the O-specific polysaccharides of common monosaccharides or disaccharide fragments. The distinctions between them stem from the presence or absence of the O-acetyl group, a different configuration of the glycosidic linkage of the N-acetylfucosamine residue and/or a different configuration at C-5 of one or both derivatives of diaminouronic acids.     

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.     

Biosynthesis and characterization of poly(3-hydroxydodecanoate) by β-oxidation inhibited mutant of Pseudomonas entomophila L48

A medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) producer Pseudomonas entomophila L48 was investigated for microbial production of 3-hydroxydodecanote homopolymer. Pseudomonas entomophila L48 was found to produce MCL PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), and 3-hydroxydodecanoate (3HDD) from related carbon sources fatty acids. In this study, some of the genes encoding key enzymes in β-oxidation cycle of P. entomophila such as 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and acetyl-CoA acetyltransferase were deleted to study the relationship between β-oxidation and PHA synthesis in P. entomophila. Among the mutants constructed, P. entomophila LAC26 accumulated over 90 wt % PHA consisting of 99 mol % 3HDD. A fed-batch fermentation process carried out in a 6 L automatic fermentor produced 7.3 g L(-1) PHA consisting of over 97 mol % 3HDD fraction. Properties of MCL PHA were significantly improved along with increasing 3HDD contents. P(2.1 mol % 3HD-co-97.9 mol % 3HDD) produced by P. entomophila LAC25 had the widest temperature range between T(g) and T(m), which were -49.3 and 82.4 °C, respectively, in all MCL PHA reported so far. The new type of PHA also represented high crystallinity caused by side-chain crystallization compared with short side chain PHA. For the first time, P(3HDD) homopolymers were obtained.     

The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain

The currently available antithrombotic agents target the interaction of platelet integrin α_IIbβ₃ (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of α_IIbβ₃ antagonists; however, the mechanisms by which α_IIbβ₃ binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for α_IIbβ₃ involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the α_IIb β-propeller domain of α_IIbβ₃ enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the α_IIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant α_IIbβ₃ receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin α_Mβ₂ exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the α_IIb β-propeller and further indicate that recognition specificity of α_IIbβ₃ for fibrin differs from that for soluble fibrinogen.     

Study of the experimental conditions for the lipase production by a newly isolated strain of Pseudomonas aeruginosa for the enantioselective hydrolysis of (±)-methyl trans-3(4-methoxyphenyl) glycidate

A lipase producing bacterium has been isolated from the soil to enantiospecifically hydrolyze the (±)-methyl trans-3(4-methoxyphenyl) glycidate (MPGM), an intermediate in the synthesis of cardiovascular drug, diltiazem. This hydrolysis provided the desired (−)-MPGM in 44% yield with 99% enantiomeric excess. The organism was identified and confirmed as Pseudomonas aeruginosa by 16S rRNA sequencing. The various physiochemical parameters have been optimized for the maximum production of lipase in shake flask. Beef extract was found to be the best nitrogen source for lipase production. The optimized cultivation conditions were 30°C with an initial medium pH 8 in shake flask. Both inoculum age and inoculum concentration have positive effect on the lipase production and (±)-MPGM (3 mM) was found to be the optimal inducer.An erratum to this article can be found at     

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

Aberrant expression of the type V collagen α1(V) chain can underlie the connective tissue disorder classic Ehlers-Danlos syndrome, and autoimmune responses against the α1(V) chain are linked to lung transplant rejection and atherosclerosis. The α1(V) collagenous COL1 domain is thought to contain greater numbers of post-translational modifications (PTMs) than do similar domains of other fibrillar collagen chains, PTMs consisting of hydroxylated prolines and lysines, the latter of which can be glycosylated. These types of PTMs can contribute to epitopes that underlie immune responses against collagens, and the high level of PTMs may contribute to the unique biological properties of the α1(V) chain. Here we use high resolution mass spectrometry to map such PTMs in bovine placental α1(V) and human recombinant pro-α1(V) procollagen chains. Findings include the locations of those PTMs that vary and those PTMs that are invariant between these α1(V) chains from widely divergent sources. Notably, an unexpectedly large number of hydroxyproline residues were mapped to the X-positions of Gly-X-Y triplets, contrary to expectations based on previous amino acid analyses of hydrolyzed α1(V) chains from various tissues. We attribute this difference to the ability of tandem mass spectrometry coupled to nanoflow chromatographic separations to detect lower-level PTM combinations with superior sensitivity and specificity. The data are consistent with the presence of a relatively large number of 3-hydroxyproline sites with less than 100% occupancy, suggesting a previously unknown mechanism for the differential modification of α1(V) chain and type V collagen properties.