Characterization of α-Glucosyltransferase from Pseudomonas mesoacidophila MX-45

α-Glucosyltransferase was purified from Pseudomonas mesoacidophila MX-45. The molecular weight was estimated to be 63,000 by SDS–PAGE, and the isoelectric point was pi 5.4. For enzyme activity based on sucrose decomposition, the optimum pH and the optimum temperature were pH 5.8 and 40°C, respectively. The ranges of stable pH and temperature were pH 5.1–6.7 and below 40°C, respectively. The purified enzyme of MX-45 converted sucrose into trehalulose (1-O-α-d-glucopyranosyl-d-fructose) and isomaltulose (palatinose, 6–O-α-d-glucopyranosyl-d-fructose) simultaneously, and the ratio of trehalulose to isomaltulose increased at lower reaction temperatures. Therefore, optimum conditions for trehalulose production were pH 5.5–6.5 at 20°C. The yield of trehalulose from sucrose (20–40% solution) was 91%. The K_m for sucrose was 19.2 ± 3.3 mm estimated by the Hanes–Woolf plot. Product inhibition was observed, and the product inhibition constant was 0.17 m. Hg²⁺, Fe³⁺, Cu²⁺, Mg²⁺, Ag⁺, Pb²⁺, glucono-1,5-lactone, and Tris(hydroxymethyl)aminomethane inhibited the reaction.     

Characterisation of Arthrobacter sp. S1 that can degrade α and β-haloalkanoic acids isolated from contaminated soil

A bacterium identified as Arthrobacter sp. S1 by 16S rRNA was isolated from contaminated soil. This is the first reported study that Arthrobacter could utilize both α-halocarboxylix acid (αHA) [2,2-dichloropropionic acid (2,2-DCP) and D,L-2-chloropropionic acid (D,L-2-CP)] and β-halocarboxylix acid (βHA) [3-chloropropionic acid (3CP)] as sole source of carbon with cell doubling times of 5?±?0.2, 7?±?0.1, and 10?±?0.1 h, respectively. More than 85 % chloride ion released was detected in the growth medium suggesting the substrates used were utilized. To identify the presence of dehalogenase gene in the microorganism, a molecular tool that included the use of oligonucleotide primers specific to microorganisms that can grow in halogenated compounds was adapted. A partial putative dehalogenase gene was determined by direct sequencing of the PCR-amplified genomic DNA of the bacterium. A comparative analysis of the deduced amino acid sequence data revealed that the amino acid sequence has a low identity of less than 15 % to both group I and group II dehalogenases, suggesting that the current putative dehalogenase amino acid sequence was completely distinct from both α-haloacids and β-haloacids dehalogenases. This investigation is useful in studying the microbial populations in order to monitor the presence of specific dehalogenase genes and to provide a better understanding of the microbial populations that are present in soil or in water systems treating halogenated compounds.     

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.     

Characterization of α-maltotetraohydrolase produced by Pseudomonas sp. MS300 isolated from the deepest site of the Mariana Trench

We have isolated a Pseudomonas-like amylase producer, the strain MS300, which displayed a large halo on starch medium, from the deepest site of the Mariana Trench. The strain MS300 produced two major and two minor α-maltotetraohydrolases (G4-amylase). The two major G4-amylases share the same molecular weight of 55 000 but had different pI values, 5.0 and 4.7, respectively. The optimum temperature for activity of both major G4-amylases is 40°C, and the optimum pH is 6.8 for one and 8.9 for the other. MS300 produced more amylase under high hydrostatic pressure than under atmospheric pressure. Strain MS300 may be active in the deep sea at a depth of 10 897 m. Received: December 11, 1997 / Accepted: April 16, 1998     

Characterization of an acid phosphatase—Glycoprotein from barley seeds

• 1.1. An acid phosphatase from barley seeds germinated for 120 hr, that is not affected by the presence of cycloheximide, is a glycoprotein.
• 2.2. The enzyme has an optimum pH 4.9 and a mol. wt of 57,000 as determined by SDS-gel electrophesis.
• 3.3. The main sugar is galactose amounting to one half of the protein and it is as either mono or disaccharide.
• 4.4. Serine and glycine predominate and hydroxyproline is absent.

     

Characterization of an α-amanitin insensitive RNA polymerase from cauliflower

α-Amanitin insensitive RNA polymerase (polymerase I isolated from apical parts of the cauliflower inflorescence was highly stable for several months at − 18°. The DEAE-cellulose fraction was more effective in utilizing denatured DNA than native DNA as a template. Optimum pH for RNA synthesis was ca 7 in the reaction mixture with Tris-HCl or with Tris-maleate buffer. From the properties examined, it seems that DNA-dependent RNA polymerase I of cauliflower differs from other eucaryotic RNA polymerases.     

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.     

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.     

Susceptibility to mercurials of clinical Pseudomonas aeruginosa isolated in México

Susceptibility to inorganic mercuric ions and to organomercurials of 237 Pseudomonas aeruginosa clinical strains isolated in Mexico was determined by agar dilution tests. Resistant strains fell into two classes: i) narrow-spectrum resistant strains (27% of total isolates) resistant only to mercuric ions and to merbromin, and most grouped in pyocin type 1; and ii) broad-spectrum resistant strains (5%) with additional resistances to thimerosal, phenylmercury, methylmercury and p-hydroxymercuribenzoate, that belonged mostly to pyocin type 10. Mercurial resistant isolates showed a higher proportion of resistance to antibiotics and metals than did mercurial sensitive isolates, and broad-spectrum resistant strains had the highest frequency of resistance to antibiotics and to tellurite and arsenate.     

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.     

Characterization of the ozone effect over an α-amylase from Bacillus licheniformis

Starch usually soils industrial process equipment, hence demanding specific washing procedures to ensure optimal products and reliable process performance. α-Amylases have been included in detergent formulations to hydrolyse starch making easier its elimination. Ozone is frequently used as disinfectant but could also help to remove the starchy soils improving the cleaning process. To study the effect of ozone on the enzyme, the ozonation of an α-amylase from Bacillus licheniformis at pH 7.5 and 25 °C was carried out. Enzyme activity assays showed that the relative α-amylase activity after ozonation decreased with increasing ozone/enzyme molar ratio exponentially. On the other hand, the ozone concentration after the reaction was negligible as some enzymatic activity remained, being the ozone consumption fast due to the high reaction kinetics of aromatic and sulfur-containing amino acids residues of the enzyme. The UV and MALDI-TOF mass spectra confirmed the oxidation of these amino acids, while the peptide bonds were unaffected. Therefore the loss of the α-amylase activity observed would be caused by the oxidation of amino acids residues directly involved in the hydrolysis mechanism such as tyrosine and histidine and/or by denaturation of the enzyme upon amino acid residues oxidation.     

Short chain α-pyrones capable of potentiating penicillin G against Pseudomonas aeruginosa

The dramatic increase in bacterial resistance over the past three decades has greatly reduced the effectiveness of nearly all clinical antibiotics, bringing infectious disease to the forefront as a dire threat to global health. To combat these infections, adjuvant therapies have emerged as a way to reactivate known antibiotics against resistant pathogens. Herein, we report the evaluation of simplified α-pyrone adjuvants capable of potentiating penicillin G against Pseudomonas aeruginosa, a Gram-negative pathogen whose multidrug-resistant strains have been labeled by the Centers for Disease Control and Prevention as a serious threat to public health.     

Characterization of PfiT/PfiA toxin–antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction

Toxin–antitoxin (TA) systems are small genetic modules usually consisting of two elements—a toxin and an antitoxin. The abundance of TA systems among various bacterial strains may indicate an important evolutionary role. Pseudomonas aeruginosa, which can be found in a variety of niches in nature, is an opportunistic pathogen for various hosts. While P. aeruginosa strains are very versatile and diverse, only a few TA systems were characterized in this species. Here, we describe a newly characterized TA system in P. aeruginosa that is encoded within the filamentous Pf4 prophage. This system, named PfiT/PfiA, is a homologue of the ParE/YefM TA system. It is a type II TA system, in which the antitoxin is a protein that binds the toxic protein and eliminates the toxic effect. PfiT/PfiA carries several typical type II characteristics. Specifically, it constitutes two small genes expressed in a single operon, PfiT inhibits growth and PfiA eliminates this effect, PfiA binds PfiT, and PfiT expression results in elongated cells. Finally, we assigned a novel function to this TA system, where an imbalance between PfiT and PfiA, favouring the toxin, resulted in cell elongation and an increase in virion production.     

Biodegradation of 2-sulfonato fatty acid methyl ester (α-SFMe): Identification of microorganisms isolated from activated sludge and their capability for degrading α-SFMe

Three bacterial strains, A, B and C, were isolated from activated sludge as 2-sulfonato-fatty-acid-methyl-ester (-SFMe)-degrading microorganisms. From the results of morphological, physiological and biochemical studies, and analyses of 16S rRNA gene sequences, isolate A was identified as Agrobacterium tumefaciens while B and C were Pseudomonas putida, respectively. To demonstrate their capability for the ultimate biodegradation of -SFMe, the degradation kinetics have been investigated using C₁₄--SFMe and 2-¹⁴C-labeled C₁₆--SFMe. The biodegradation was determined by measuring dissolved organic carbon (DOC) and released SO₄ ^2–, in the shake-culture test, and evolved ¹⁴CO₂ in the modified Organisation for Economic Co-operation and Development (OECD) test. In the shake culture test with C₁₄--SFMe, DOC removal was progressive throughout the test. Liberation of inorganic sulfate started after DOC removal and then rapidly increased. During the ¹⁴CO₂ evolution tests, the mineralization of radiolabeled carbon started quickly and reached about 80% of the initially added radioactivity at the end of the tests. The results obtained indicated that all of the isolates had the capability for ultimately degrading -SFMe through the oxidation of the alkyl carbons and desulfonation (cleavage of the C-S linkage).     

Amino acid sequences of allophycocyanin α- and β-subunits isolated from Anabaena cylindrica: Presence of an unknown derivative of aspartic acid in the β-subunit

Allophycocyanin (APC) α- and β-subunits were isolated from Anabaena cylindrica and their amino acid sequences were determined. The α- and β-subunits consisted of 160 and 161 amino acid residues, respectively, and lacked tryptophan. The β-subunit contained an unknown derivative of aspartic acid. The sequences were compared with those of other allophycocyanins.     

R-(−)-mandelic acid production from racemic mandelic acids by Pseudomonas polycolor with asymmetric degrading activity

The microbial asymmetric degradation of S-(+)-mandelic acid was investigated in order to develop a practical process for R-(−)-mandelic acid production from racemic mandelic acids. Among the 790 culture strains tested, microorganisms belonging to the Brevibacterium, Pseudomonas, Rhodococcus, Rhodotorula, Rhodosporidium, Sporobolomyces and Gibberella genera exhibited high S-(+)-mandelic acid degrading activity. Pseudomonas polycolor IFO 3918 was determined to be the best strain and used as a biocatalyst for eliminating the S-(+)-isomer. The maximum rate of S-(+)-isomer degradation was obtained at 30°C and pH 7.0. Under these optimal conditions, the S-(+)-isomer in a racemic mandelic acid 45 g/l mixture was completely degraded within 24 h, with 20 g of R-(−)-mandelic acid per liter remaining in the reaction mixture. Crystalline R-(−)-mandelic acid with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess was obtained at a yield of 35% by acidification of the reaction mixture, extraction with ethyl acetate and subsequent concentration.     

Characterization of an exo-acting intracellular α-amylase from the hyperthermophilic bacterium Thermotoga neapolitana

We cloned and expressed the gene for an intracellular α-amylase, designated AmyB, from the hyperthermophilic bacterium Thermotoga neapolitana in Escherichia coli. The putative intracellular amylolytic enzyme contained four regions that are highly conserved among glycoside hydrolase family (GH) 13 α-amylases. AmyB exhibited maximum activity at pH 6.5 and 75°C, and its thermostability was slightly enhanced by Ca²⁺. However, Ca²⁺ was not required for the activity of AmyB as EDTA had no effect on enzyme activity. AmyB hydrolyzed the typical substrates for α-amylase, including soluble starch, amylose, amylopectin, and glycogen, to liberate maltose and minor amount of glucose. The hydrolytic pattern of AmyB is most similar to those of maltogenic amylases (EC 3.2.1.133) among GH 13 α-amylases; however, it can be distinguished by its inability to hydrolyze pullulan and β-cyclodextrin. AmyB enzymatic activity was negligible when acarbose, a maltotetraose analog in which a maltose residue at the nonreducing end was replaced by acarviosine, was present, indicating that AmyB cleaves maltose units from the nonreducing end of maltooligosaccharides. These results indicate that AmyB is a new type exo-acting intracellular α-amylase possessing distinct characteristics that distinguish it from typical α-amylase and cyclodextrin-/pullulan-hydrolyzing enzymes.     

The Origin of tRNA Deduced from Pseudomonas aeruginosa 5′ Anticodon-Stem Sequence

Origins of Life and Evolution of Biospheres - The riddle of the origin of life is unsolved as yet. One of the best ways to solve the riddle would be to find a vestige of the first life from...     

First partial synthesis of α-boswellic acid from oleanolic acid

While β-boswellic acid is very readily available by extraction from frankincense resin, the accessibility of α-boswellic from the resin involved great effort and tedious purification procedures. Alternatively, a partial synthesis from readily available oleanolic acid was developed, the key steps of which are a reduction of the carboxyl group C-28 furnishing a methyl group, followed by palladium-assisted oxidation of the methyl group C-24, and configurational inversion at C-3.