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.     

Production,Purification and Crystallization of 3α-Hydroxysteroid Dehydrogenase of Pseudomonas putida

The ability of formation of 3α-hydroxysteroid dehydrogenase was studied in bacteria and actinomycetes. The enzyme activity was found in several bacteria belonging to the genera Pseudomonas, Bacillus and Corynebacterium, when they were grown on cholic acid as a sole source of carbon. Of these bacteria, Pseudomonas putida NRRL B–11064 isolated from soil, showed the highest activity of 3α-hydroxysteroid dehydrogenase. The enzyme was purified from the cell-free extract by procedure including fractionation with ammonium sulfate and column chromatographies on DEAE-celluIose, Sephadex G–100 and hydroxylapatite. Crystals of the enzyme were obtained by the addition of ammonium sulfate to the purified enzyme in the presence of glycerol or polyethylene glycol. The overall purification was about 550-fold with an yield of 18.5%. The crystalline enzyme was homogeneous on polyacrylamide disc electrophoresis and analytical ultracentrifugation (s_20,w=3.2).     

Comparative biochemistry of α-glucan-utilization in pseudomonas amyloderamosa and Pseudomonas saccharophila

Growth patterns on and utilization of various α-glucans were investigated in Pseudomonas amyloderamosa and P. saccharophila. Maltose, maltodextrins (average chain length 7 glucosyl units) and glycogen supported excellent growth of both organisms and were extensively metabolized, although glycogen utilization in P. saccharophila was preceded by a prolonged lag phase. P. amyloderamosa produced limited growth on amylopectin and the carbohydrate was only partly degraded. It seemed likely that many of the unit chains liberated from amylopectin had a length exceeding the substrate range accepted by the maltodextrin permease (transport) system. A correlation was established between the pH of the medium and the utilization of glycogen and amylopectin for growth in P. amyloderamosa. The carbohydrates were at least partly utilizable at pH 6.0, whereas they could not support any growth at pH 6.5. Most likely, the lack of growth at the higher pH reflected the low activity of isoamylase at this pH. The enzyme patterns of maltodextrin catabolism in the two bacteria were established. Intracellularly, maltodextrin phosphorylase and 4-α-glucanotransferase occurred in both. Degradation of extracellular α-glucans was mediated by a mainly intracellular isoamylase in P. amyloderamosa, whereas P. saccharophila possessed an extracellular α-amylase and a firmly cell-bound pullulanase.     

Cephalosporinase and penicillinase activities of a β-lactamase from Pseudomonas pyocyanea

1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.     

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far. Received: 24 May 1999 / Accepted: 7 October 1999     

Molecular Cloning and Characterization of γ-Glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694

γ-Glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694 (PnGGT) exhibited higher hydrolytic activity than transfer activity, as compared with other γ-glutamyltranspeptidases (GGTs). PnGGT showed little activity towards most of L-amino acids and towards glycyl-glycine, which is often used as a standard γ-glutamyl accepter in GGT transfer reactions. The preferred substrates for PnGGT as a γ-glutamyl accepter were amines such as methylamine, ethylamine, and isopropylamine.     

Assimilation of γ-butyrobetaine,and d-and l-carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

The metabolic pattern of utilization of [1,2,3,4-¹⁴C, methyl-³H] -butyrobetaine and d-and l-[1-¹⁴C, methyl-³H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on -butyrobetaine, Ps. putida rapidly metabolized -butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or -butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that -butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or -butyrobetaine showed no detectable -butyrobetaine hydroxylase activity.     

Crystal structure analysis and enzymatic characterization of γ-glutamyltranspeptidase from Pseudomonas nitroreducens

Theanine (γ-glutamylethylamide) is an amino acid analog that reduces blood pressure and improves immune responses. The ?-glutamyltranspeptidase (GGT) from Pseudomonas nitroreducens IFO12694 (PnGGT) has a unique preference for primary amines as ?-glutamyl acceptors over standard L-amino acids and peptides. This characteristic is useful for the synthesis of theanine. We used X-ray crystallographic analysis to understand the structural basis of PnGGT’s hydrolysis and transpeptidation reactions and to characterize its previously unidentified acceptor site. Structural studies of PnGGT have shown that key interactions between three residues (Trp385, Phe417, and Trp525) distinguish PnGGT from other GGTs. We studied the roles of these residues in the distinct biochemical properties of PnGGT using site-directed mutagenesis. All mutants showed a significant decrease in hydrolysis activity and an increase in transpeptidase activity, suggesting that the aromatic side chains of Trp385, Phe417, and Trp525 were involved in the recognition of acceptor substrates.

Abbreviations: ?-glutamyl peptide, theanine, X-ray crystallography.     

Variation of resistance and infectivity between Pseudomonas fluorescens SBW25 and bacteriophage Ф2 and its therapeutic implications

<正>Dear Editor,Studies of the coevolutionary dynamics between Pseudomonas fluorescens SBW25 and bacteriophageФ2 can explore host resistance and parasite infectivity with applications in the ecological and therapeutic fields.Coevolutionary dynamics determine the efficacy of phage-based therapy.In the study described here,bacterial resistance and phage infectivity fluctuated with culture     

Purification and characterization of an exo-type β-N-acetylglucosaminidase from Pseudomonas fluorescens JK-0412

Aims: To purify and characterize an exo‐acting chitinolytic enzyme produced from a Gram‐negative bacterium Pseudomonas fluorescens JK‐0412. Methods and Results: A chitinolytic bacterial strain that showed confluent growth on a minimal medium containing powder chitin as the sole carbon source was isolated and identified based on a 16S ribosomal DNA sequence analysis and named Ps. fluorescens JK‐0412. From the culture filtrates of this strain, a chito‐oligosaccharides‐degrading enzyme was purified to apparent homogeneity with a molecular mass of 50 kDa on SDS–PAGE gels. The kinetics, optimum pH and temperature, and substrate specificity of the purified enzyme (named as NagA) were determined. Conclusions: An extracellular chitinolytic enzyme was purified from the Ps. fluorescens JK‐0412 and shown to be an exo‐type β‐N‐acetylglucosaminidase yielding GlcNAc as the final product from the natural chito‐oligosaccharides, (GlcNAc)_n, n = 2–5. Significance and Impact of the Study: As NagA is secreted extracellularly in the presence of colloidal chitin, Ps. fluorescens JK‐0412 can be recognized as a potent producer for industry‐level and cost‐effective production of chitinolytic enzyme. This enzyme appears to have potential applications as an efficient tool for the degradation of chitinous materials and industry‐level production of GlcNAc. To the best of our knowledge, this is the first report on an exo‐type chitinolytic enzyme of Pseudomonas species.     

Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Partial Purification and Some Properties of Mitomycin C Inactivating Factors of Pseudomonas convexa 4–87

Strains producing higher levels of cellulolytic enzymes were selected from among 520 strains of plant pathogenic fungi, Fusarium species, and F. oxysporum strain SUF850 was found to be the best producer. When strain SUF850 was cultured using one of three polysaccharides, Avicel, carboxy- methyl cellulose (CMC) or xylan, as a carbon source, the culture filtrate contained degrading activi- ties toward all three substrates, i.e., irrespective of the carbon source used. From the culture filtrate of Avicel-grown cells, four distinct enzymes were purified to homogeneity, as judged on SDS-PAGE. They were designated as CMCase I, CMCase II, /Miitrophenyl-β-d-cellobiosidase and xylanase, and the characteristics of the individual enzymes were examined and compared.     

Diagnostic utility of combination of inducer and inhibitor based assay in detection of Pseudomonas aeruginosa producing AmpC β-lactamase

The diagnostic utility of inducer and inhibitor based assays among 214 AmpC positive isolates of Pseudomonas aeruginosa were evaluated. Of different methods, combination of ceftazidime-imipenem antagonism and boronic acid inhibition tests came up with maximum sensitivity (76%) and specificity (100%). This combination showed reliability for both inducible and non-inducible AmpC producers.     

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.     

Population dynamics of a dual Pseudomonas putida–Pseudomonas fluorescens biofilm in a capillary bioreactor

Most biofilm studies employ single species, yet in nature biofilms exist as mixed cultures, with inevitable effects on growth and development of each species present. To investigate how related species of bacteria interact in biofilms, two Pseudomonas spp., Pseudomonas fluorescens and Pseudomonas putida, were cultured in capillary bioreactors and their growth measured by confocal microscopy and cell counting. When inoculated in pure culture, both bacteria formed healthy biofilms within 72?h with uniform coverage of the surface. However, when the bioreactors were inoculated with both bacteria simultaneously, P. putida was completely dominant after 48?h. Even when the inoculation by P. putida was delayed for 24?h, P. fluorescens was eliminated from the capillary within 48?h. It is proposed that production of the lipopeptide putisolvin by P. putida is the likely reason for the reduction of P. fluorescens. Putisolvin biosynthesis in the dual-species biofilm was confirmed by mass spectrometry.     

Gene cloning, structural gene and promoter identification, and active assay of the phosphatidylcholine synthase of Pseudomonas sp. strain 593

Pseudomonas sp. strain 593, a soil bacterium, is able to use exogenous choline to synthesize phosphatidylcholine via phosphatidylcholine synthase (Pcs). A 2020 bp DNA fragment that hybridized to a Pcs probe was cloned. This fragment contained a large open reading frame (ORF) with two potential ATG start sites that would encode for 293 and 231 amino acid proteins. Fragments containing the two ORFs encoded Pcs when they were inserted into the expression vector pET23a and expressed under the control of the T7 promoter in Escherichia coli BL21(DE3) pLysS. However, when the two ORFs were inserted into the cloning vector pMD18-T and expressed without control of the plasmid promoter in E. coli DH5α, only the larger clone exhibited Pcs activity. This suggested that the larger fragment contained a native promoter driving expression of the smaller ORF. A promoter activity assay, in which DNA fragments were inserted into the promoter-probe plasmid pCB182 and β-galactosidase activity of E. coli transformants was tested, demonstrated that a promoter is indeed present in the DNA region. All results together indicate that the 696 bp ORF, not the larger 897 bp ORF, encodes the Pcs in Pseudomonas sp. strain 593 and carries a promoter in front of its 5' terminus.     

Purification and biochemical characterization of a detergent stable α-amylase from Pseudomonas stutzeri AS22

This study reports the purification and biochemical characterization of a novel maltotetraose-forming-α-amylase from Pseudomonas stutzeri AS22, designated PSA. The P. stutzeri α-amylase (PSA) was purified from the culture supernatant to homogeneity by Sepharose mono Q anion exchange chromatography, ultrafiltration and Sephadex G-100 gel filtration, with a 37.32-fold increase in specific activity, and 31% recovery. PSA showed a molecular weight of approximately 57 kDa by SDS-PAGE. The N-terminal amino acid sequence of the first 7 amino acids was DQAGKSP. This enzyme exhibited maximum activity at pH 8.0 and 55°C, performed stably over a broad range of pH 5.0 ≈ 12.0, but rapidly lost activity above 50°C. Both potato starch and Ca²⁺ ions have a protective effect on the thermal stability of PSA. The enzyme activity was inhibited by Hg²⁺, Mn²⁺, Cd²⁺, Cu²⁺, and Co²⁺, and enhanced by Ba²⁺. PSA belonged to the EDTA-sensitive α-amylase. The purified enzyme showed high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), and oxidizing agents, such as sodium per borate and H₂O₂. In addition, PSA showed excellent compatibility with a wide range of commercial solid and liquid detergents at 30°C, suggesting potential application in the detergent industry. Maltotetraose was the specific end product obtained after hydrolysis of starch by the enzyme for an extended period of time, and was not further degraded.     

l-Alanine-α-Ketobutyrate Aminotransferase of Pseudomonas sp.

Modified fungal product 4-O-methylascochlorin (MAC) is an experimental agent affecting lipid and carbohydrate metabolism in mammals. The hypocholesterolemic properties of MAC were studied using rats fed on a standard laboratory diet. Because of the insolubility in water, reproducibility of the hypocholesterolemic activity had usually been poor for rats fed ad libitum. The difficulty was overcome by controlled reverse-phase feeding; MAC significantly lowered serum total cholesterol (s-TC) in rats only when given by gastric intubation soon after diet intake.

MAC increased fecal excretion of neutral and acidic sterols and also increased biliary flow accompanying increments in biliary cholesterol, bile acids and phospholipids. A much larger increase in neutral sterols was characteristic for MAC. However, intestinal absorption of cholesterol and cholic acid was unaffected by MAC. Three mechanisms therefore seemed to be working in hypocholesterolemic activity: (a) withdrawal of hepatic cholesterol into bile, (b) a larger fecal loss of sterols following increment of biliary sterols and (c) enhanced bile acid synthesis compensating the larger fecal loss. A negative sterol balance often leads to an increase in hepatic cholesterogenesis. However, cholesterogenesis, as judged from incorporation of the precursors, was unchanged by MAC.