Effect of high hydrostatic pressure on Escherichia coli and Pseudomonas fluorescens strains in ovine milk

Ovine milk that had been standardized to 6% fat was inoculated with Escherichia coli 405 CECT and Pseudomonas fluorescens 378 CECT at a rate of 10(6) and 10(7) cfu/ml, respectively, and treated with high hydrostatic pressure. Treatments consisted of combinations of pressure (300, 400, 450, and 500 MPa), temperature (2, 10, 25, and 50 degrees C), and time (5, 10, and 15 min). Inactivation (> 6 log cfu/ml) of both strains was observed at 50 degrees C for all pressures and treatment times. A similar level of inactivation occurred at > or = 450 MPa and 25 degrees C for E. coli and at > or = 400 MPa and 10 degrees C for P. fluorescens. Destruction was lowest at 10 degrees C for E. coli and at 25 degrees C for P. fluorescens. The test strain of E. coli was more baroresistance than was the P. fluorescens strain.     

Aluminum detoxication mechanism in Pseudomonas fluorescens is dependent on iron

The soil microbe Pseudomonas fluorescens has been shown to detoxify aluminum by the elaboration of a soluble metabolite where the trivalent metal is sequestered [Appanna and St. Pierre, FEMS Microbiol. Lett. 24 (1994) 327-332]. The inclusion of 5 mM iron in the growth medium elicited an entirely disparate detoxification strategy. In this instance, the two trivalent metals were immobilized in a gelatinous lipid-rich residue. Dialysis and ultracentrifugation studies indicated that the test metals were being transformed from early stages of growth and were associated with phosphatidylethanolamine. However, at 45 h of cellular multiplication, most of the metals were deposited as an insoluble residue. X-ray fluorescence analyses identified the constituents of this mineral essentially as aluminum, iron and phosphorus. Scanning electron microscopy and energy dispersive X-ray microanalysis of the dialysate, isolated at 35 h of microbial growth, revealed thread-like structures associated with nodule-like bodies that were rich in the two test metals. Transmission electron microscopic studies aided in the visualization of iron and aluminum inclusions within the bacterial cells.     

Primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

The amino acid sequence of the p-hydroxybenzoate hydroxylase (4-hydroxybenzoate,NADPH:oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) monomer from Pseudomonas fluorescens has been determined. The sequence was elucidated by a combination of the results from an X-ray crystallographic study at 0.25 nm resolution (Wierenga, R.K., de Jong, R.J., Kalk, K.H., Hol, W.G.J. and Drenth, J. (1979) J. Mol. Biol. 131, 55-73) and from protein sequence analysis. The polypeptide chain of the monomer contains 394 amino acids and has a molecular weight of 44 299.     

Outbreak of Pseudomonas fluorescens bacteremia among oncology patients

From 7 to 24 March 1997, four patients developed Pseudomonas fluorescens bacteremia at the hospital; one on the oncology ward and the other three in the chemotherapy room. These patients all had underlying malignancies and had the Port-A-Cath (Smiths Industries Medical Systems, Deltec, Inc., St. Paul, Minn.) implants. Three patients had primary bacteremia, and one had Port-A-Cath-related infection. None of these patients had received a blood transfusion before the episodes of bacteremia. All patients recovered: two received antimicrobial agents with in vitro activity against the isolates, and the other two did not have any antibiotic treatment. A total of eight blood isolates were recovered from these patients during the febrile episodes that occurred several minutes after the infusion of chemotherapeutic agents via the Port-A-Cath. These isolates were initially identified as P. fluorescens or Pseudomonas putida (four), Burkholderia (Ralstonia) pickettii (three), and a non-glucose-fermenting gram-negative bacillus (one) by routine biochemical methods and the Vitek GNI card. These isolates were later identified as P. fluorescens on the basis of the characteristic cellular fatty acid chromatogram and the results of supplemental biochemical tests. The identification of identical antibiotypes by the E test and the random amplified polymorphic DNA patterns generated by arbitrarily primed PCR of the isolates showed that the outbreak was caused by a single clone of P. fluorescens. Surveillance cultures of the possibly contaminated infusion fluids and disinfectants, which were performed 7 days after recognition of the last infected patient, failed to isolate P. fluorescens. This report of a small outbreak caused by P. fluorescens suggests that timely, accurate identification of unusual nosocomial pathogens is crucial for early initiation of an epidemiological investigation and timely control of an outbreak.     

Cellular response to a multiple-metal stress in Pseudomonas fluorescens

Pseudomonas fluorescens multiplied in a minimal mineral medium supplemented with millimolar amounts of aluminum (5 mM), iron (5 mM), zinc (3 mM), calcium (2 mM) and gallium (1 mM). A slight decrease in growth rate and a 22% diminution in cellular yield were observed as compared to the control medium. Citrate, the sole source of carbon to which the test metals were complexed, was completely utilized. Although at stationary phase of growth most of the metals were immobilized in an exocellular lipid-rich residue, ultracentrifugation and dialysis studies revealed that metals were associated with phosphatidylethanolamine (PE) from early stages of growth. As growth progressed the metal content of the soluble cellular extract increased reaching an optimum at 35 h of incubation. However, no detectable amounts of metals in this cellular component were discerned at stationary phase of growth. There appeared to be no marked variation in exocellular protein and carbohydrate production in control and metal-stressed cultures. Transmission electron microscopic studies revealed metal rich bodies associated with the cytoplasm. Scanning electron microscopic analyses of the dialyzate aided in the identification of the metal-rich bodies associated with elongated structures comprised of carbon, oxygen and phosphorus. PE appeared to be an important organic constituent of the gelatinous residue.     

Specificity of substrate recognition by Pseudomonas fluorescens N3 dioxygenase. The role of the oxidation potential and molecular geometry

Pseudomonas fluorescens N3 is able to grow on naphthalene as the sole carbon and energy source. The mutant TTC1, blocked at the dihydrodiol dehydrogenase level, which can transform the hydrocarbon into the corresponding dihydrodiol, has been used to produce bioconversion products. To rationalize the different grades of conversion obtained with different substrates, a study was performed using non-naphthalene derivatives, including benzenes, conjugated benzenes, and polycyclic aromatic hydrocarbons. The corresponding diols obtained by bioconversion have been isolated and characterized. A theoretical model that considers both energy and geometry factors has been proposed to rationalize the experimental data. Good agreement has been found between the calculated values and the experimental results.     

Effect of protein content on low temperature inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F

The formation of cholic acid and chenodeoxycholic acid through cleavage of the side chains of CoA esters of 3alpha,7alpha,12alpha-trihydroxy-5beta-choles tan-26-oic acid and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid is believed to occur in peroxisomes. Recently, we found a new peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and suggested that this bifunctional protein is responsible for the conversion of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-2 4-en-26-oyl-CoA and 3alpha,7alpha-dihydroxy-5beta-cholest-24-en-26-oyl-CoA to their 24-oxo-forms. In the present study, the products of this bifunctional protein reaction were analyzed by gas chromatography-mass spectrometry, and the formation of 24-oxo-27-nor-cholestanes was confirmed. Previously, we found a new thiolase in Caenorhabditis elegans, P-44, and suggested that P-44 and sterol carrier protein x, a peroxisomal protein, constitute a second group of 3-oxoacyl-CoA thiolases. The production of cholic acid and chenodeoxycholic acid from the precursors on incubation with the bifunctional protein and sterol carrier protein x or P-44 was confirmed by gas chromatography.     

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.     

The structure of the exopolysaccharide of Pseudomonas fluorescens strain H13

PURPOSE: Behavioral dysfunction of memory process arising 4 months after whole brain irradiation (30 Gy/10 fractions/12 days) has been demonstrated in 16-27 month old rats, as compared with non irradiated rats. This study was therefore aimed at delivering the same irradiation in young rats and comparing results with those previously obtained in old rats. MATERIAL AND METHODS: Thirty-three 4-month old rats were included into the study. Eighteen received whole brain irradiation (30 Gy/10 fractions/12 days), and 18 were given sham irradiation. Sequential behavior studies were done before irradiation and during the 7 months following irradiation. RESULTS: Significant decrease in memory function was observed in irradiated rats 1 month (p < 0.001), 3 months (p < 0.013), and 6 months (p = 0.007) post-irradiation. This was accompanied by learning deficit 1 month (p = 0.01), 4.5 months (p = 0.03), and 7 months (p = 0.009) post-irradiation. CONCLUSION: Response to radiation therapy observed in young rats differed from that observed in old rats. Young rats showed earlier decrease in memory function than old rats, but this deficit was followed by partial recovery. Learning deficits also arised earlier in young rats than in old rats. In two cases this deficit was permanent.     

Molecular cloning and analysis of a lipase gene from Pseudomonas fluorescens No. 33

The gene encoding an extracellular lipase from Pseudomonas fluorescens No. 33 was cloned and sequenced. A single open reading frame consisting of 1,428 nucleotides that encoded a mature protein of 476 amino acids was recognized. Sequence analysis showed that the deduced molecular weight of 50,209 agreed with the molecular weight of the purified lipase as measured by SDS-PAGE and the lipase lacked a signal peptide. The presence of a repeating motif, GXXGXDXXX, suggested that the lipase might be exported and secreted via a system that involves the ATP-binding cassette protein.     

Biodegradation of benzene, toluene, ethylbenzene, and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (> 1000 mg l-1) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l-1. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Cloning, sequence, and properties of the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens

The gene encoding the soluble pyridine nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli. STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues. The gene is highly similar to an E. coli gene of unknown function.     

Effect of charcoal-broiled beef on phenacetin metabolism in man

When charcoal-broiled beef was fed to human volunteers, who were then given phenacetin orally, the concentration of phenacetin in the plasma was lowered, but its half-life in the plasma was not changed. The data suggest that feeding charcoal-broiled beef enhances the metabolism of orally administered phenacetin in the intestine or during its first pass through the liver, or both.     

c-type cytochromes and manganese oxidation in Pseudomonas putida MnB1

Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides. We used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and we characterized some of these mutants. The mutants were divided into three groups: mutants defective in the biogenesis of c-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan. The mutants in the first two groups were cytochrome c oxidase negative and did not contain c-type cytochromes. Mn(II) oxidation capability could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation.     

Influence of natural antioxidants on in vitro lipoprotein oxidation

Susceptibility of low-density lipoproteins (LDL) to oxidation might be a critical risk factor in the development and progression of atherosclerosis. The oxidation involves the degradation of polyunsaturated fatty acids, the formation of lysolecithin, oxysterols and aldehyde modification of lysine residues on Apo B100. The oxidation products have a number of biological activities such as cytotoxicity, atherogenesis, and carcinogenesis. The aim of this study was to investigate the in vitro antioxidant effects of vitamins E, A, and C on LDL. LDL was isolated from plasma by density gradient high-speed centrifugation and used as 0.1 microM/l isotonic solution. LDL oxidation was triggered by simple incubation with Cu2+ (1, 2, 5, 10, 12, 20 microM/l), in absence or presence of lipid-soluble or water-soluble antioxidants in different concentrations (tocopherols--0.5, 1, 2, and 4 microM/l; cerotenoids--0.1, 0.2, and 0.4 microM/l; ascorbate--2.5, 5, and 10 microM/l). The LDL oxidability was measured by continuous spectrophotometrical monitoring at 234 nm of the increased formation of conjugated diene hydroperoxides. The oxidation curves showed a profile with an inhibition period followed by a propagation period and were quantitatively characterized by two parameters: lag-phase (expressed in minutes), and propagation rate (expressed in changes of absorbance--delta E234nm/min). Lag-phase--the period of inhibition oxidation--was attributed to the ability of LDL (by antioxidants) to resist oxidation in vitro. LDL lag-phase decreased and propagation rate increased with the increasing of copper concentration. In conclusion: 1) susceptibility of LDL to oxidation depends on both the concentration of pro-oxidant stimuli and the entity and concentrations of antioxidants; 2) antioxidants retard the process through which LDL undergo oxidation in vitro when exposed to copper ions; 3) a synergistic effect may also be involved, as water-soluble vitamin C keeps the fat-soluble vitamin E and vitamin A within LDL.     

Regulation of asparaginase and glutaminase activity in Pseudomonas fluorescens mutants lacking in the ability to synthesize glutamic acid

A mutant of Pseudomonas fluorescens, AG-M49, has been isolated, which lacks the ability to synthesize glutamic acid. The mutant contains both aspartase A and aspartase AG. The mutant was characterized by a considerably high activity of glutaminase and aspartase, increasing with the rate flow, during its continuous cultivation on a medium with L-glutamate. L-aspartate penetrates the cells and induces formation of the both deamidases in the mutant. In these conditions, the concentration of exocellular ammonia increases. Asparagine, but not glutamine, also stimulates production of the deamidases in the mutant.     

The diversity of lipases from psychrotrophic strains of Pseudomonas: a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens

Strains of Pseudomonas fluorescens and Ps. fragi are the predominant psychrotrophs found in raw milk and may cause spoilage due to the secretion of hydrolytic enzymes such as lipase and protease. The diversity of lipases has been examined in Pseudomonas isolates from raw milk which represent different taxonomic groups (phenons). Significant diversity was found using both DNA hybridization and immunoblotting techniques, which has implications for the development of a diagnostic test. The lipase-encoding gene (lipA) was cloned from one strain, C9, of Ps. fluorescens biovar V. In contrast to previously reported lipase sequences from Ps. fluorescens, the gene encodes a lipase of M(r) 33 kDa. Alignment of all known Pseudomonas and Burkholderia lipase amino acid sequences indicates the existence of two major groups, one of M(r) approximately 30 kDa comprising sequences from Ps. fragi, Ps. aeruginosa, Ps. fluorescens C9 and Burkholderia, and one of approximately 50 kDa comprising Ps. fluorescens lipases. The lipase from C9 does not contain a signal peptide and is presumed to be secreted via a signal peptide-independent pathway. The lipA gene of strain C9 was disrupted by insertional mutagenesis. The mutant retained its lipolytic phenotype, strongly suggesting the presence of a second lipase in this strain.     

The amino-acid sequence of the three smallest CNBr peptides from p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: (Formula: See Text).