Ps. fluorescens

Proc Natl Acad Sci U S A, 1989 Oct, 86(19), 7378 - 81
gamma-Aminobutyric acid uptake by a bacterial system with neurotransmitter binding characteristics; Guthrie GD et al.; gamma-Aminobutyric acid (GABA), an amino acid, has been found in every class of living organisms . In higher organisms, GABA is a neurotransmitter and binds with high affinity and specificity to GABA receptors on neurons in a sodium-independent reaction that is saturable . The role of GABA in organisms lacking nervous tissue is not known . This report describes, in a strain of Pseudomonas fluorescens, a GABA uptake system with binding characteristics like those of the GABA (type A) brain receptor . The binding was saturable and specific for GABA, was sodium-independent, was of high affinity (Km = 65 nM), and was inhibited competitively by muscimol, a potent GABA analogue . The bacterial GABA system included a homogeneous binding site, and no cooperative interaction was found between sites . To our knowledge, such a system for GABA, or other neurotransmitters, in a bacterium has not been reported.

Mol Microbiol, 1989 Sep, 3(9), 1211 - 9
Conserved serine-rich sequences in xylanase and cellulase from Pseudomonas fluorescens subspecies cellulosa: internal signal sequence and unusual protein processing; Hall J et al.; The complete nucleotide sequence of the xynA gene coding for a xylanase (XYLA) expressed by Pseudomonas fluorescens subspecies cellulosa, has been determined . The structural gene consists of an open reading frame of 1833 bp followed by a TAA stop codon . Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid sequence with that derived by N-terminal analysis of purified forms of the xylanase . The signal peptide present at the N terminus of mature XYLA closely resembles signal peptides of other secreted proteins . Truncated forms of the xylanase gene, in which the sequence encoding the N-terminal signal peptide had been deleted, still expressed coli . XYLA contains domains which are homologous to an endoglucanase expressed by the same organism . These structures include serine-rich sequences . Bal31 deletions of xynA revealed the extent to which these conserved sequences, in XYLA, were essential for xylanase activity . Downstream of the TAA stop codon is a G + C-rich region of dyad symmetry (delta G = 24 kcal) characteristic of E . coli Rho-independent transcription terminators.

Biochem Biophys Res Commun, 1989 Aug 30, 163(1), 49 - 55
Liver microsomes contain two distinct NADPH-Monooxygenases with NH2-terminal segments homologous to the flavin containing NADPH-monooxygenase of Pseudomonas fluorescens; Ozols J; Two NADPH-reductase preparations (FAD-containing monooxygenases) were isolated from rabbit liver microsomes, referred to as from 1 and from 2 . Purification was achieved by means of anion-exchange, cation-exchange and hydroxylapatite chromatography in the presence of cholate and Nonidet P-40 . Affinity chromatography on 2', 5'-ADP Sepharose was used to increase the purity and to concentrate the enzyme . On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, form 1 exhibited a single band at Mr 58,500 and form 2 at Mr 58,000 . The NH2- terminus of form 1 is blocked, whereas the NH2-terminus of form 2 is homologous to the NADPH-phydroxybenzoate hydrolase from Pseudomonas fluorescens . The latter and the form 2 enzyme share 11 identical residues in the NH2-terminal segment of 15 residues . Both forms were subjected to tryptic cleavages and peptide mapping . Sequence analysis of the peptides obtained indicated that forms 1 and 2 are similar but not identical proteins . A tryptic peptide, homologous to residues 3 to 32 of form 2 enzyme was isolated from the form 1 protein . This segment has 24 residues that are identical to the form 2 and contains the consensus sequence Gly-X-Gly-X-X-Gly, found in most FAD binding proteins . These results indicate that the NADPH-monooxygenase system consists of at least two distinct proteins representing different gene products.

Appl Environ Microbiol, 1989 Aug, 55(8), 1949 - 54
Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads; Huisman GW et al.; The fluorescent pseudomonads are classified as a group, one characteristic of which is that they do not accumulate poly-3-hydroxybutyrate (PHB) during nutrient starvation in the presence of excess carbon source . In this paper we show that prototype strains from this subclass, such as Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens, do accumulate poly-3-hydroxyalkanoates (PHA) when grown on fatty acids . These PHAs are composed of medium-chain-length (C6 to C12) 3-hydroxy fatty acids . The ability to form these polyesters does not depend on the presence of plasmids . A specificity profile of the enzymes involved in the biosynthesis of PHA was determined by growing Pseudomonas oleovorans on fatty acids ranging from C4 to C18 . In all cases, PHAs were formed which contained C6 to C12 3-hydroxy fatty acids, with a strong preference for 3-hydroxyoctanoate when Ceven fatty acids were supplied and 3-hydroxynonanoate when Codd fatty acids were the substrate . These results indicate that the formation of PHAs depends on a specific enzyme system which is distinct from that responsible for the synthesis of PHB . While the fluorescent pseudomonads are characterized by their inability to make PHB, they appear to share the capacity to produce PHAs . This characteristic may be helpful in classifying pseudomonads . It may also be useful in the optimization of PHA production for biopolymer applications.

Can J Microbiol, 1989 Jul, 35(7), 675 - 80
Survival of and plasmid stability in Pseudomonas and Klebsiella spp . introduced into agricultural drainage water; Trevors JT et al.; Cell survival and plasmid stability in Pseudomonas fluorescens R2f and Pseudomonas putida CYM 318 containing respectively, plasmid RP4 and pRK2501, and Klebsiella aerogenes NCTC 418 harboring plasmid pBR322 were studied in sterile and nonsterile agricultural drainage water under both aerobic and anaerobic conditions and in the absence and presence of added nutrients . Both Pseudomonas strains survived well in sterile drainage water incubated aerobically, with or without added nutrients . However, Klebsiella aerogenes NCTC 418 (pBR322) only survived in the presence of added nutrients . Pseudomonas fluorescens R2f (RP4) and K . aerogenes NCTC 418 (pBR322) did not survive under anerobic conditions without added nutrients, but showed good survival in the presence of nutrients . Survival of all three strains was negatively affected in nonsterile agricultural drainage water when compared with survival in sterile water . Maintenance of the three plasmids was host, plasmid, and environment dependent . Plasmid pBR322 was not stably maintained in K . aerogenes NCTC 418 under all conditions used in the study, and pRK2501 was readily lost from P . putida CYM 318 . Maintenance of RP4 by P . fluorescens R2f was markedly influenced by added nutrients, which caused a loss of the plasmid from cells . The results of the present study demonstrate the influence of nutrients, O2, and native microorganisms on the survival of introduced bacterial strains and plasmid stability in agricultural drainage water.

J Hosp Infect, 1989 Jul, 14(1), 73 - 8
Emission of viable bacteria in the exhaust flue gases from a hospital incinerator; Blenkharn JI et al.; The exhaust gases from an oil-fired hospital waste incinerator were examined during normal incinerator operation . The design-specified operating temperature was 800 degrees C in the primary combustion chamber and 1000 degrees C in the secondary chamber . Flue gas temperatures, measured from the sampling point at the base of the exhaust stack, varied over the range 186-305 degrees C, and bacteria were recovered from this position in numbers up to 400 cfu m-3 (mean 56 cfu m-3) . No sampling was performed at the top of the stack where flue gases were discharged to the atmosphere . Isolates were predominantly gram positive, i.e . Bacillus spp., coagulase negative staphylococci and Staphylococcus aureus, although low numbers of gram negative species (Pseudomonas fluorescens and other pseudomonads) were also recovered . Our results suggest that incineration may not constitute an absolute method of sterilization for clinical waste.

J Gen Microbiol, 1989 Jul, 135 ( Pt 7), 1787 - 97
Molecular cloning and sequence determination of the lpd gene encoding lipoamide dehydrogenase from Pseudomonas fluorescens; Benen JA et al.; The lpd gene encoding lipoamide dehydrogenase (dihydrolipoamide dehydrogenase; EC 1.8.1.4) was isolated from a library of Pseudomonas fluorescens DNA cloned in Escherichia coli TG2 by use of serum raised against lipoamide dehydrogenase from Azotobacter vinelandii . Large amounts (up to 15% of total cellular protein) of the P . fluorescens lipoamide dehydrogenase were produced by the E . coli clone harbouring plasmid pCJB94 with the lipoamide dehydrogenase gene . The enzyme was purified to homogeneity by a three-step procedure . The gene was subcloned from plasmid pCJB94 and the complete nucleotide sequence of the subcloned fragment (3610 bp) was determined . The derived amino acid sequence of P . fluorescens lipoamide dehydrogenase showed 84% and 42% homology when compared to the amino acid sequences of lipoamide dehydrogenase from A . vinelandii and E . coli, respectively . The lpd gene of P . fluorescens is clustered in the genome with genes for the other components of the 2-oxoglutarate dehydrogenase complex.

Wei Sheng Wu Xue Bao, 1989 Jun, 29(3), 155 - 60
{A sarcoma-static new species of Pseudomonas, Pseudomonas jinanensis sp . nov.}; Cai MY et al.; A strain of Gram negative bacteria was isolated from the surface soil of Wuying Hill at Jinan, Shandong province with Gause's medium in 1973 . It is a strain of antagonistic bacteria for hysterocervicoma, hepatoma and melanoma of mice screened from 2100 strains of bacteria . It is also antagonistic to Staphylococcus aureus, Bacillus subtilis and Micrococcus . It is a Gram negative bacterium with lophotrichous polar flagella . Straight rods in shape or with a little slightly curved rods, 0.5-0.6 X 1-2 microns, randomly arranged, poly-beta-hydroxybutyrate granules are accumulated in cells after 2-5 days cultivation . Water green soluble pigment and green fluorescent pigment are produced . Respiratory metabolism, chemoorganotroph, many carbon-containing organic compounds can be used as carbon sources, such as glucose, trehalose, ethanol, cellulobiose, fucose, arginine and betaine, but propionic acid or tartaric acid is not utilized . Inorganic nitrogen containing compounds can be used ae the sole source of nitrogen . No growth factor is necessary for growth . Gelatin is hydrolyzed . Starch and cellulose are not hydrolyzed . Nitrate is not reduced . Arginine dihydrolase is produced . Levan is produced from sucrose . Growth occurs from 7 degrees C to 37 degrees C and from pH 5.65-8.40 . No growth occurs at 40 degrees C and at pH value below 4.86 . It can not grow autotrophically with hydrogen . Its G + C contents in DNA is 58.1 mol% . DNA-DNA hybridization experiments reveals a relatedness value of 58.6% between this strain and Ps . fluorescens . The above evidence shows that this strain differs from all species known in Pseudomonas, such as Pseudomonas fluorescens group . Pseudomonas caryophylli, Pseudomonas cepacia, Pseudomonas marginata, Pseudomonas acidovorans, Pseudomonas testosteroni and Pseudomonas delafieldii.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1989 Jun, 55(6), 1640 - 1
Rapid detection and identification of Legionella pneumophila by a membrane immunoassay; Berube A et al.; Legionella pneumophila was detected and identified by an immunoblot assay using a monoclonal antibody specific to serogroups 1 to 8 . Samples containing L . pneumophila were plated on buffered charcoal yeast extract agar supplemented with glycine, vancomycin, and polymyxin B . After incubation at 35 degrees C for 3 days, colonies were transferred to nitrocellulose membranes by blotting . Simultaneous detection and identification of L . pneumophila were done by treating the membrane with the monoclonal antibody and a peroxidase conjugate to mouse immunoglobulins . A diffuse cross-reaction was observed with Pseudomonas fluorescens colonies, but this was a low-level reaction that could easily be differentiated from the strong specific reactions to L . pneumophila.

Appl Environ Microbiol, 1989 Jun, 55(6), 1578 - 83
Evaluation of different approaches for identification of xenobiotic- degrading pseudomonads; Busse H et al.; Different approaches were evaluated to identify aerobic, gram-negative, biodegradative isolates assumed to be pseudomonads . Quinone and polyamine analysis allowed rapid identification to the genus level, i.e., allocation of those isolates belonging to the Pseudomonas fluorescens complex which represents the phylogenetically defined core of the heterogeneous genus Pseudomonas . Subsequent studies concentrated only on these true pseudomonads . The multiple-test system API 20NE, determination of the moles percent G+C content, and polyacrylamide gel electrophoresis of soluble proteins aided in identification on the species level . Polyacrylamide gel electrophoresis of both soluble proteins and whole-cell lipopolysaccharides allowed recognition of identical strains and double isolates, which were confirmed by DNA-DNA hybridization.

FEMS Microbiol Lett, 1989 Jun, 50(3), 337 - 43
pEG plasmid involved in styrene degradation: molecular dimorphism and integration of a segment into the chromosome; Ruzzi M et al.; In the Pseudomonas fluorescens strain ST the ability to utilize styrene as the sole carbon source is due to the presence of a plasmid, pEG . In the present report we show that pEG contains two inverted repeat sequences and we present evidence indicating that the catabolic genes are localized in these repeats . The region separating the inverted repeats can assume alternative orientations . In the chromosome of strain ST, a 3 kbp region is homologous to sequences present at one end of the plasmid repeats . This region is present in one copy in the chromosome and could be a specific site for integration of the plasmid . We suggest that this sequence, which is present twice in the pEG plasmid and once in the chromosome, might be a transposon-like element.

Biochemistry, 1989 May 2, 28(9), 3935 - 9
A time-resolved fluorescence study of azurin and metalloazurin derivatives; Hutnik CM et al.; Nickel and cobalt derivatives of Pseudomonas fluorescens (ATCC 13525) azurin were prepared and their steady-state fluorescence and time-resolved fluorescence monitored . Like the copper-containing native protein, the fluorescence decay of both metallo derivatives was best fit to a sum of three exponentials, whereas the apoazurin from which they were prepared obeyed single-exponential decay kinetics . However, comparison of the lifetimes and fractional of each of the components in these derivatives to those in the oxidized and reduced native proteins revealed significant differences . These results suggest that the presence of a metal center in azurin imparts a conformational heterogeneity which is strongly dependent on the nature of the metal center . Further, the results are used to comment on current ideas concerning the geometry of the active site in this redox protein.

Biochemistry, 1989 May 2, 28(9), 3923 - 34
Confirmation that multiexponential fluorescence decay behavior of holoazurin originates from conformational heterogeneity; Hutnik CM et al.; Homologous azurins from Pseudomonas fluorescens (ATCC 13525) and Pseudomonas aeruginosa (ATCC 10145) were examined by a number of electrophoretic techniques, and their copper to protein stoichiometry was determined by atomic absorption and amino acid analysis . Provided that the spectral ratio (A620/A280 or A625/A280) was 0.53 and there was no evidence of a Soret band in the absorption spectrum, then these criteria can be used to judge the homogeneity of the azurin sample . If the spectral ratio was less than 0.50, evidence suggested a nonreconstitutable, non-trypsin-digestible apoazurin was present . The fluorescence decay of these homogeneous holoazurins included three components, not two as previously reported {Szabo, A . G., et al . (1983) Biophys . J . 41, 233-244} . Whereas the decay times were nearly the same for the azurins from the different sources, the fractional fluorescence of each component varied with the azurin measured . The fluorescence of the corresponding apoazurins, prepared by a refined procedure, obeyed monoexponential decay kinetics . The temperature and pH effects on the fluorescence behavior of these homologous azurins are presented with the pH study suggesting an influence by a group which titrates between pH 5 and pH 7 . When taken together these results confirm that the multiexponential decay behavior originates from conformational heterogeneity and not from contamination by an apo form.

J Bacteriol, 1989 May, 171(5), 2819 - 26
Cloning and characterization of a gene encoding an outer membrane protein required for siderophore-mediated uptake of Fe3+ in Pseudomonas putida WCS358; Marugg JD et al.; In iron-limited environments plant-growth-stimulating Pseudomonas putida WCS358 produces a yellow-green fluorescent siderophore called pseudobactin 358 . Ferric pseudobactin 358 is efficiently taken up by cells of WCS358 but not by cells of another rhizophere-colonizing strain, Pseudomonas fluorescens WCS374 . A gene bank containing partial Sau3A DNA fragments from WCS358 was constructed in a derivative of the broad-host-range cosmid pLAFR1 . By mobilization of this gene bank to strain WCS374 a cosmid clone, pMR, which made WCS374 competent for the utilization of pseudobactin 358 was identified . By subcloning of the 29.4-kilobase (kb) insert of pMR the essential genetic information was localized on a BglII fragment of 5.3 kb . Tn5 mutagenesis limited the responsible gene to a region of approximately 2.5 kb within this fragment . Since the gene encodes an outer membrane protein with a predicted molecular mass of 90,000 daltons, it probably functions as the receptor for ferric pseudobactin 358 . The gene is flanked by pseudobactin 358 biosynthesis genes on both sides and is on a separate transcriptional unit . WCS374 cells carrying pMR derivatives with Tn5 insertions in the putative receptor gene did not produce the 90,000-dalton protein anymore and were unable to take up Fe3+ via pseudobactin 358 . In WCS358 cells as well as in WCS374 cells the gene is expressed only under iron-limited conditions.

J Bacteriol, 1989 May, 171(5), 2401 - 5
Benzaldehyde lyase, a novel thiamine PPi-requiring enzyme, from Pseudomonas fluorescens biovar I; Gonzalez B et al.; Pseudomonas fluorescens biovar I can grow on benzoin as the sole carbon and energy source . This ability is due to benzaldehyde lyase, a new type of enzyme that irreversibly cleaves the acyloin linkage of benzoin, producing two molecules of benzaldehyde . Benzaldehyde lyase was purified 70-fold and found to require catalytic amounts of thiamine PPi (TPP) and a divalent cation as cofactors . Optimal activity was obtained with a 1.0 mM concentration of Mn2+, Mg2+, or Ca2+ . Gel permeation chromatography indicated a native molecular weight of 80,000, whereas the enzyme migrated in sodium dodecyl sulfate-containing polyacrylamide gels as a single polypeptide with a molecular weight of 53,000 . Benzaldehyde lyase is highly specific; of a variety of structurally related compounds tested, only benzoin and anisoin (4,4'-dimethoxybenzoin) acted as substrates, their apparent Kms being 9.0 x 10(-3) and 3.25 x 10(-2) mM, respectively . A catalytic mechanism for the enzyme is proposed.

J Microencapsul, 1989 Apr-Jun, 6(2), 165 - 76
Immobilization of enzyme by microencapsulation and application of the encapsulated enzyme in the catalysis; Iso M et al.; Microencapsulation of lipase (Pseudomonas fluorescens) was carried out using (W/O)/W two-phase emulsion technique . Polystyrene (PS) and Styrene-Butadiene Rubber (SBR) were utilized as wall materials either separately or in mixture . A particular composition of 2:1 PS-SBR yielded homogeneous and tough wall structure, resilient to the impact and tight confinement of enzyme macromolecules . Performance of the encapsulated enzyme was evaluated employing the hydrolysis of triacetin (triglyceride of acetic acid) as a model substrate of the enzyme catalysis . A mathematical model was developed to simulate the behaviour of hydrolysis, which was derived under the assumption that the diffusion of small molecules (substrate and products) through the wall of microcapsules plays a dominant role to the reaction rate . Inhibition of the reaction by the decreasing pH due to the release of acetic acid was also taken into account . The calculated values agreed quite well with the observed data.

J Biol Chem, 1989 Mar 15, 264(8), 4715 - 21
Human aldehyde dehydrogenase . Purification and characterization of a third isozyme with low Km for gamma-aminobutyraldehyde; Kurys G et al.; An enzyme which catalyzes dehydrogenation of gamma-aminobutyraldehyde has been purified to homogeneity from human liver and identified as an isozyme of aldehyde dehydrogenase (EC 1.2.1.3); two other isozymes, previously obtained in a homogeneous form, are known as E1 and E2 . Affinity chromatography on NAD-agarose (N6 with 8 carbon spacer) yields homogeneous enzyme which migrates as two components on isoelectric focusing with pI = 5.3 and 5.45 . These two components, separated by fast protein liquid chromatography on a Mono-P HR 5/20 column, have similar Km values for gamma-aminobutyraldehyde, acetaldehyde, propionaldehyde, and NAD . The Km value for gamma-aminobutyraldehyde is 8.0-14.0 microM versus 760 microM for E1 and 512 microM for E2 . The enzyme's molecular weight, subunit molecular weight, and amino acid composition are similar to those of the E1 and E2 isozymes . The enzyme also interacts with anti-E1 and anti-E2 antibodies; it is relatively insensitive to disulfiram inhibition and is neither activated nor inhibited by magnesium . Its absorption spectrum, where the ratio of 280/260 nm is 1.1 and a weak absorption is seen in the 340 nm range (Racker band), suggests the presence of bound coenzyme . gamma-Aminobutyraldehyde dehydrogenase (with Km value of 15 microM for gamma-aminobutyraldehyde) was previously partially purified from Pseudomonas fluorescens (Jakoby, W.B., and Fredericks, J . (1959) J . Biol . Chem . 234, 2145-2150) but never from a mammalian organism.

Mikrobiologiia, 1989 Mar-Apr, 58(2), 229 - 35
{Fatty acid composition of lipid A in Pseudomonas fluorescens}; Veremeichenko SN et al.; The fatty acid composition of lipid A was studied using gas-liquid chromatography (GLC) and GLC-mass spectrometry in Pseudomonas fluorescens strains of biovars A, B, C, i, F and G, the type strain ATCC 13525 (biovar A) inclusive . The following fatty acids were identified as predominant in the composition of lipid A in the strains representing biovars A, B, C, i, F and G: 3-hydroxydecanoic (3-OH C10:0), 2-hydroxydodecanoic (2-OH C12:0), 3-hydroxydodecanoic (3-OH C12:0), dodecanoic (C12:0), hexadecanoic (C16:0), octadecanoic (C18:0), hexadecenoic (C16:1) and octadecenoic (C18:1) acids . Lipid A of a biovar G strain differed noticeably from other strains in its fatty acid composition . Its main components were as follows: 3-hydroxytetradecanoic (3-OH C14:0), 3-hydroxypentadecanoic (3-OH C15:0) and dodecanoic (C12:0) fatty acids . The coefficients of similarity were determined for lipid A specimens isolated from the studied strains of P . fluorescens by calculating their fatty acid composition with a computer.

J Appl Bacteriol, 1989 Mar, 66(3), 227 - 33
Partial immunological characterization of heat-stable proteinases from Pseudomonas spp . of dairy origin; Azcona JI et al.; A homogeneous extracellular heat-stable proteinase from Pseudomonas fluorescens AH-70 was used to prepare antiserum in rabbits . The rabbit antiserum was used to study the antigenic relationship of the proteinases from 26 psychrotrophic Pseudomonas spp . isolated from raw milk . The inhibition of the proteinases by the antiserum, the gel precipitin reactions and the use of a double-antibody sandwich ELISA, revealed that proteinase AH-70 is immunologically related to proteinases from 8/26 other Pseudomonas strains . These results also indicate that the immunological techniques for the detection of proteolytic enzymes in raw milk require antibody preparations of broader specificity.

J Bacteriol, 1989 Feb, 171(2), 887 - 95
Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis; Binnie C et al.; The anhydrotetracycline (ATC) oxygenase enzyme which carries out the conversion of ATC to dehydrotetracycline was purified and the N-terminal amino acid sequence was determined . The sequence displays a significant similarity to that of the p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens . This is consistent with the activity of the oxygenase, i.e., addition of a hydroxyl moiety to an aromatic ring structure . Oligonucleotide probes were designed and used to clone the corresponding fragment of chromosomal DNA from Streptomyces rimosus . This DNA fragment was used to screen a cosmid library, allowing the isolation of flanking DNA sequences . Surprisingly, the gene was located within the previously cloned cluster of genes involved in the synthesis of the biosynthetic intermediate ATC and not as had been expected (P . M . Rhodes, N . Winskill, E . J . Friend, and M . Warren, J . Gen . Microbiol . 124:329-338, 1981) at a separate locus on the other side of the chromosome . Subcloning of an appropriate DNA fragment from one of the cosmid clones onto pIJ916 produced Streptomyces lividans transformants which synthesized oxytetracycline.

Antimicrob Agents Chemother, 1989 Feb, 33(2), 156 - 63
Mechanism of mupirocin transport into sensitive and resistant bacteria; Capobianco JO et al.; Pseudomonic acid A (mupirocin) blocks protein synthesis in bacteria by inhibition of bacterial isoleucyl-tRNA synthetase . {16, 17-3H}mupirocin, isolated from a methionine auxotroph of Pseudomonas fluorescens, was used to study transport of this antibiotic into sensitive and resistant strains of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli . The transport of mupirocin into sensitive bacteria was energy independent and temperature dependent (decreased uptake at lower temperatures), indicating non-carrier-mediated passive diffusion . Uptake was also saturable with time or increasing antibiotic concentration . The saturable intracellular binding site, most likely the target isoleucyl-tRNA synthetase as determined by the amount of bound mupirocin (2,700 to 3,100 molecules per cell), caused concentration of the antibiotic within the cell . E . coli transformed with a plasmid containing ileS overproduced the target enzyme and demonstrated greater accumulation of mupirocin than a strain containing a control plasmid . The concentrations needed to half saturate (Kd) these binding sites in B . subtilis and S . aureus were 35 and 7 nM, respectively . In gram-positive organisms trained for mupirocin resistance, uptake was not saturable with increasing antibiotic concentration, and intra- and extracellular concentrations of drug equilibrated with time . Kinetic analysis of crude isoleucyl-tRNA synthetase from trained and untrained B . subtilis strains revealed differences in apparent Ki for mupirocin (resistant strain SB23T, Ki = 71.1 nM; sensitive strain SB23, Ki = 33.5 nM), while the Km for isoleucine remained unchanged (2.7 to 2.9 microM) . A Km of 0.4 micromolar isoleucine and Ki of 24 nM mupirocin was demonstrated for isoleucyl-tRNA synthetase from sensitive S . aureus 730a, while no isoleucyl-tRNA synthetase activity was detected in extracts of resistance-trained S . aureus 3000 even at 40 micromolar isoleucine, suggesting instability of the enzyme . Free isoleucine pools differed between sensitive (0.26 micromolar) and resistance-trained (1.06 micromolar) S . aureus . Our results demonstrate that (i) mupirocin enters cells by passive diffusion, (ii) mupirocin concentrates in sensitive bacteria due to binding to isoleucyl-tRNA synthetase, and (iii) resistance to mupirocin involves restricted access to the binding site of isoleucyl-tRNA synthetase.

Eur J Biochem, 1989 Feb 1, 179(2), 307 - 14
The temperature and pH dependence of some properties of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens; Van Berkel WJ et al.; The free and complexed flavoprotein, p-hydroxybenzoate hydroxylase, was studied by light-absorption, circular-dichroism and fluorescence techniques as a function of the pH . The following compounds served as ligands for the enzyme: p-hydroxybenzoate, p-fluorobenzoate, benzoate, p-aminobenzoate and tetrafluoro-p-hydroxybenzoate . Depending on the technique used, the various ligands exhibit pH-dependent physical properties and dissociation constants . The data can be fitted with pKa values in the range 7.7-7.9 . It is suggested that this pKa value belongs to a tyrosine residue in the active center of the enzyme . This assignment is supported by published data and additional experiments.

Mikrobiologiia, 1989 Jan-Feb, 58(1), 54 - 9
{The esterase activity of a pigmented bacterial group belonging to the Pseudomonas genus}; Imshenetskii AA et al.; Bacteria belonging to the Pseudomonas genus and isolated from zonal soils in different geographical zones of the USSR as well as from the rhizosphere of cultivated and wild plants were tested for their esterase activity . The studied collection of cultures included 205 strains of different pigmented Pseudomonas species which, according to the conventional taxonomy, were assigned to the so-called "Pseudomonas fluorescens complex" . As was shown in this study, many Pseudomonas species are potential producers of nonspecific esterases . P . maltophilia and P . geniculata synthesizing pyomelanin have the highest activity of esterase . The activity of esterase correlates with the formation of a melanin-like pigment in Pseudomonas cultures . It also correlates with the species to which a culture belongs, which makes it possible to use this property as an additional criterion for the identification of Pseudomonas species.

Folia Microbiol (Praha), 1989, 34(6), 515 - 24
Metabolic characteristics of bacterial cells entrapped in beaded calcium alginate and/or pectate gels; Toth D et al.; A mixture of heterotrophic bacteria and collection strains of Escherichia coli and Pseudomonas fluorescens were immobilized in calcium alginate or pectate gels . Comparison of respiratory activity, substrate uptake and biosynthetic capacity of immobilized cells showed that both types of carriers permit a prolonged preservation of metabolic activity but the transfer of substances through the gel is faster in the pectate . Morphological changes include some intracellular structures, partial shrinkage of the plasma membrane of immobilized cells, and transformation of a rod-like cell shape to an oval one.

Nucleic Acids Symp Ser, 1989, (21), 3 - 4
Enzyme catalyzed acylation and deacylation of the sugar moieties of nucleosides; Nozaki K et al.; We have found that a lipase from Pseudomonas fluorescens (PFL) accelerated regioselective acylation of 2'-deoxynucleosides with the use of acid anhydrides as acyl donor in dry polar solvents . Different regioselective deacylation of 3',5'-di-O-acyl-2'-deoxynucleosides was found to take place when a lipase (PFL) or a protease from Bacillus subtilis (Subtilisin) was used.

Med Parazitol (Mosk), 1989 Jan-Feb, (1), 35 - 40
{Mixed infections in the pathology of blood-sucking larvae pathology . 2 . Entomopathogenic properties of bacterioviral complexes}; Mikhnovskaia ND et al.; Mixed infection of the mosquitoes' larvae of the first age group by densonucleasis virus and entomopathogenic strains of Pseudomonas fluorescens and Serratia marcescens enhanced viral infection in the presence of toxicosis induced by exogenic entomotoxic bacterial metabolites . Possibility of interaction between bacterial cells and the mosquito densonucleasis virus, producing an adverse effect on the duration of the disease, was demonstrated . Duration of the disease provoked by bacterioviral infection of the larvae was of a specific character: nontypical degeneration and untimely replacement of intestinal epithelium at the fourth larval stage were observed along with acute viral lesions of all the tissues.

Biol Met, 1989, 2(1), 18 - 24
Ferripyoverdine-reductase activity in Pseudomonas fluorescens; Halle F et al.; Enzymatic release of iron from ferripyoverdine through a reductive mechanism was demonstrated in cell-free extracts of Pseudomonas fluorescens . Ferripyoverdine reductase activity was localized primarily in the cytoplasm and/or periplasm and appeared not to be affected by the iron status of the cells . The reaction required a strict anaerobic environment and was fully inhibited by oxygen, whereas NADH was the most effective reductant . Ferripyoverdines from other bacterial sources (P . aeruginosa ATCC 15692, P . fluorescens ATCC 13525, P . fluorescens ATCC 17400) were able to serve as iron sources as well as ferric citrate . However, the activity with ferric citrate was not strongly affected by oxygen and did not display the characteristic lag phase observed with ferripyoverdines, suggesting the occurrence of a specific ferric citrate iron reductase . FMN should play a critical role in the reductive mechanism since it was absolutely required for the activity to occur with an intensively dialyzed cell-free extract, whereas it greatly stimulated (50-fold) the NADH-mediated activity of a crude extract.

Microbios, 1989, 60(242), 59 - 61
In vitro antibacterial effect of the essential oil of Thymus longiflorus Boiss; Cruz T et al.; The essential oil of Thymus longiflorus Boiss was tested for its in vitro antibacterial activity . The results showed antibacterial effects against Gram-positive and Gram-negative bacteria, especially against Pseudomonas fluorescens and Mycobacterium phlei.

J Chromatogr, 1988 Dec 29, 434(1), 31 - 41
Highly sensitive determination and characterization of intact cellular ester-linked phospholipids using liquid chromatography-plasma spray mass spectrometry; Odham G et al.; Liquid chromatographic class separations of common cellular phospholipids combined with plasma spray ionization of the effluents were investigated . Comparison with true thermospray ionization involving ammonium acetate buffering revealed a gain in total ionization in the plasma spray of a factor of approximately 10 using a cation-exchange column and a solvent mixture consisting of acetonitrile-methanol-water (400:100:15, v/v) . Plasma spray ionization studies of bovine brain polyphosphoinositides interrelated by the phosphate content in the inositol moiety showed almost identical monoglyceride and diglyceride ion clusters, indicating possibilities of studying the biochemical turnover of such phospholipids . Plasma spray ionization liquid chromatography-mass spectrometry of bacterial membrane phospholipids (Pseudomonas fluorescens) revealed possibilities of obtaining indications of individual fatty acid compositions from the spectra of the phosphatidylinositol and phosphatidylethanolamine fractions present . Conventional gas chromatographic fatty acid analysis agreed with the direct mass spectrometric structure elucidations . Interestingly, the two phospholipid classes had different relative fatty acid compositions with a significantly higher degree of cyclic fatty acids in the phosphatidyl ethanolamines . Plasma spray ionization yielded linear dose-response curves for both the monoglyceride and diglyceride fragment signals in the selected-ion monitoring mode . The detection limit for the monoglyceride and diglyceride species of phosphatidylcholine under the chromatographic and mass spectrometric conditions used was found to be in the picogram range.

Acta Crystallogr C, 1988 Dec 15, 44 ( Pt 12), 2220 - 2
Structure of the pseudomonad fungal antibiotic phenazine-1-carboxylic acid; Jones GP et al.; C13H8N2O2, Mr = 224.2, monoclinic, Cc, a = 3.955 (1), b = 19.278 (4), c = 13.468 (1) A, beta = 98.90 (2) degrees, V = 1015 (2) A3, Z = 4, D chi = 1.468 Mg m-3, lambda (Mo K alpha) = 0.7107 A, mu = 0.061 mm-1, F(000) = 464, T = 293 (2) K, R = 0.047 for 571 observed reflections . The crystal-structure determination of the title compound, a phenazine antibiotic from Pseudomonas fluorescens 2-79 (NRRL B-15132), confirms its structure as phenazine-1-carboxylic acid . The molecular packing is described by discrete stacks of molecules parallel to the a axis with the distance between the essentially planar molecules being ca 3.96 A; there are no significant intermolecular contacts in the lattice.

J Gen Microbiol, 1988 Dec, 134 ( Pt 12), 3239 - 47
Molecular cloning of multiple xylanase genes from Pseudomonas fluorescens subsp . cellulosa; Gilbert HJ et al.; Pseudomonas fluorescens subsp . cellulosa was shown to express extracellular xylanases . Genes encoding these enzymes were isolated from a gene library of P . fluorescens subsp . cellulosa DNA, constructed in bacteriophage lambda 47.1 . One of the phages (PXC) that expressed xylanase also conferred the ability to hydrolyse carboxymethylcellulose . An 11.8 kb HindIII DNA restriction fragment and a 6.2 kb EcoRI DNA fragment were subcloned from two distinct xylanase-expressing phages, into pUC18, to yield recombinant plasmids pGHJ4 and pGHJ5 respectively . Cells of Escherichia coli harbouring either of these two plasmids, or plasmid pJHH1 (comprising the cellulase gene from PXC, previously cloned on a 7.3 kb partial EcoRI DNA fragment in pUC18), expressed xylanase activity . The positions of the xylanase genes in the recombinant plasmids were elucidated by subcloning and transposon mutagenesis . In pJHH1 the xylanase gene was adjacent to the DNA region encoding the endoglucanase . The polysaccharide-degrading genes in pJHH1 were transcribed from different promotors . Hybridization studies revealed that the xylanase genes encoded by pGHJ4 and pGHJ5 showed strong homology . All three cloned enzymes cleaved p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-cellobioside . Xylan and glucose did not affect expression of xylanase in E . coli strains harbouring pJHH1, pGHJ4 or pGHJ5.

J Bacteriol, 1988 Oct, 170(10), 4865 - 73
Specificity of pyoverdine-mediated iron uptake among fluorescent Pseudomonas strains; Hohnadel D et al.; Pyoverdine-mediated iron transport was determined for seven fluorescent Pseudomonas strains belonging to different species . For all strains, cell or cell outer membrane and iron(III)-pyoverdine combinations were compared with their homologous counterparts in uptake, binding, and cross-feeding experiments . For four strains (Pseudomonas putida ATCC 12633, Pseudomonas fluorescens W, P . fluorescens ATCC 17400, and Pseudomonas tolaasii NCPPB 2192), the pyoverdine-mediated iron transport appeared to be strictly strain specific; pyoverdine-facilitated iron uptake by iron-starved cells and binding of ferripyoverdine to the purified outer membranes of such cells were efficient only in the case of the homologous systems . Cross-feeding assays, in liquid or solid cultures, resulted, however, especially for P . fluorescens ATCC 17400, in some discrepancies compared with uptake and binding assays, suggesting that growth experiments are the least likely to yield correct information on specificity of the pyoverdine-mediated iron transport . For the three other strains (P . fluorescens ATCC 13525, P . chlororaphis ATCC 9446, and P . aeruginosa ATCC 15692), cross-reactivity was demonstrated by the uptake, binding, and cross-feeding experiments . In an attempt to determine which parts of the iron transport system were responsible for the specificity, the differences in amino acid composition of the pyoverdines, together with the differences observed at the level of the iron-sensitive outer membrane protein pattern of the seven strains, are discussed.

Appl Environ Microbiol, 1988 Oct, 54(10), 2578 - 9
Metabolism of volatile chlorinated aliphatic hydrocarbons by Pseudomonas fluorescens; Vandenbergh PA et al.; A Pseudomonas fluorescens strain designated PFL12 was isolated from soil and water that were contaminated with various chloroaliphatic hydrocarbons . The isolate was able to metabolize 1,2-dichloroethane, 1,1,2-trichloroethane, 1,2-dichloropropane, 2,2-dichloropropane, and trichloroethylene.

Appl Environ Microbiol, 1988 Oct, 54(10), 2432 - 8
Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems; Compeau G et al.; The fate of spontaneous chromosomal rifampin-resistant (Rifr) mutants of Pseudomonas putida and Pseudomonas fluorescens in sterile and live organic soil from which they were isolated was studied . In sterile native-soil assays, a Rifr mutant of P . putida showed no decrease in competitive fitness when compared with the wild-type parent . However, mutants of P . fluorescens were of two general categories . Group 1 showed no difference from the wild type in terms of growth rate, competitive fitness, and membrane protein composition . Group 2 showed a slower growth rate in both minimal and enriched media and an altered membrane protein profile . These mutants also demonstrated decreased competitive fitness compared with the wild-type strain . In live soil, the Rifr P . putida strain persisted throughout the 38-day test period with a decay rate of 0.7 log10 CFU/g of soil per 10 days . A group 1 Rifr P . fluorescens mutant maintained its inoculated titer for 7 to 10 days and then decayed at a rate of 0.2 to 0.4 log10 CFU/g of soil per 10 days . A group 2 Rifr P . fluorescens mutant remained at its titer for 1 to 5 days before decaying at a two- to threefold-faster rate . These findings indicate that rifampin resistance may not be an innocuous mutation in some pseudomonads and that marked strains should be compared with wild-type parents before being used as monitors of parental strain survival . Colonization of sterile soil with either the wild-type or mutant strain precluded normal colonization of the second added strain.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1988 Oct, 170(10), 4748 - 56
Molecular cloning of a Pseudomonas syringae pv . syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants; Huang HC et al.; A cosmid clone isolated from a genomic library of Pseudomonas syringae pv . syringae 61 restored to all Tn5 mutants of this strain studied the ability to elicit the hypersensitive response (HR) in tobacco . Cosmid pHIR11 also enabled Escherichia coli TB1 to elicit an HR-like reaction when high levels of inoculum (10(9) cells per ml) were infiltrated into tobacco leaves . The cosmid, which contains a 31-kilobase DNA insert, was mobilized by triparental matings into Pseudomonas fluorescens 55 (a nonpathogen that normally causes no plant reactions), P . syringae pv . syringae 226 (a tomato pathogen that causes the HR in tobacco), and P . syringae pv . tabaci (a tobacco pathogen that causes the HR in tomato) . The plant reaction phenotypes of all of the transconjugants were altered . P . fluorescens(pHIR11) caused the HR in tobacco and tomato leaves and stimulated an apparent proton influx in suspension-cultured tobacco cells that was indistinguishable from the proton influx caused by incompatible pathogenic pseudomonads . P . syringae pv . tabaci(pHIR11) and P . syringae pv . syringae 226(pHIR11) elicited the HR rather than disease symptoms on their respective hosts and were no longer pathogenic . pHIR11 was mutagenized with TnphoA (Tn5 IS50L::phoA) . One randomly chosen mutant, pHIR11-18, no longer conferred the HR phenotype to P . fluorescens . The mutation was marker-exchanged into the genomes of P . syringae pv . syringae strains 61 and 226 . The TnphoA insertions in the two pseudomonads abolished their ability to elicit any plant reactions in all plants tested . The results indicate that a relatively small portion of the P . syringae genome is sufficient for the elicitation of plant reactions.

Eur J Biochem, 1988 Sep 15, 176(2), 449 - 59
Chemical modification of tyrosine-38 in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by 5'-p-fluorosulfonylbenzoyladenosine: a probe for the elucidation of the NADPH binding site? Involvement in catalysis, assignment in sequence and fitting to the tertiary structure; van Berkel WJ et al.; p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens was covalently modified by the nucleotide analog 5'-(p-fluorosulfonylbenzoyl)-adenosine in the presence of 20% dimethylsulfoxide . The inactivation reaction is pH-dependent and does not obey pseudo-first-order kinetics, due to spontaneous hydrolysis of the reagent . The kinetic data further indicate that a weak, reversible enzyme-inhibitor complex is an intermediate in the inactivation reaction and that only one amino acid residue is responsible for the loss of activity . The inactivation is strongly inhibited by NADPH and 2',5'ADP . Steady-state kinetics and 2',5'ADP bioaffinity chromatography of the modified enzyme suggest that the essential residue is not directly involved in NADPH binding . Sequence studies show that Tyr-38 is the main residue protected from modification in the presence of NADPH . From crystallographic studies it is known that the hydroxyl group of Tyr-38 is 1.84 nm away from the active site . Model-building studies using computer graphics show that this distance can be accommodated when FSO2BzAdo binds in an extended conformation with the sulfonylbenzoyl portion in an orientation different from the nicotin-amide ring of NADPH.

J Bacteriol, 1988 Aug, 170(8), 3499 - 508
Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var . tritici; Thomashow LS et al.; Pseudomonas fluorescens 2-79 (NRRL B-15132) and its rifampin-resistant derivative 2-79RN10 are suppressive to take-all, a major root disease of wheat caused by Gaeumannomyces graminis var . tritici . Strain 2-79 produces the antibiotic phenazine-1-carboxylate, which is active in vitro against G . graminis var . tritici and other fungal root pathogens . Mutants defective in phenazine synthesis (Phz-) were generated by Tn5 insertion and then compared with the parental strain to determine the importance of the antibiotic in take-all suppression on wheat roots . Six independent, prototrophic Phz- mutants were noninhibitory to G . graminis var . tritici in vitro and provided significantly less control of take-all than strain 2-79 on wheat seedlings . Antibiotic synthesis, fungal inhibition in vitro, and suppression of take-all on wheat were coordinately restored in two mutants complemented with cloned DNA from a 2-79 genomic library . These mutants contained Tn5 insertions in adjacent EcoRI fragments in the 2-79 genome, and the restriction maps of the region flanking the insertions and the complementary DNA were colinear . These results indicate that sequences required for phenazine production were present in the cloned DNA and support the importance of the phenazine antibiotic in disease suppression in the rhizosphere.

Biochim Biophys Acta, 1988 Jul 13, 950(2), 204 - 14
Characterization and expression in Escherichia coli of an endoglucanase gene of Pseudomonas fluorescens subsp . cellulosa; Lejeune A et al.; An endoglucanase gene of Pseudomonas fluorescens subsp . cellulosa present on plasmid pRUCL150 and expressed in Escherichia coli was subcloned in plasmid pBR322 . Plasmid pRUCL153 contained the smallest DNA insert (2.9 kb) with endoglucanase activity . The plasmids directed the synthesis of a mostly periplasmic enzyme in E . coli and the level of enzyme activity was comparable in several strains . Analysis by non-denaturing polyacrylamide gel electrophoresis of the endoglucanase produced with various recombinant plasmids showed that it was unique . The endoglucanase gene on plasmid pRUCL153 was localized by physical mapping of independent transposon Tn5 insertions . Hence, its size was estimated to be approx . 1.3 kb . In vivo radioactive labelling of plasmid-encoded proteins using minicells, followed by denaturing polyacrylamide gel electrophoresis, allowed us to determine the size of the endoglucanase: Mr 40,000 for the precursor and Mr 38,000 for the mature enzyme . It was demonstrated that no cellulase operon, but a single gene, was cloned . The direction of transcription of the gene was determined by placing it under the control of the promoter of the lactose operon.

Prikl Biokhim Mikrobiol, 1988 Jul-Aug, 24(4), 493 - 8
{Transformation of 2,4,6-trinitrotoluene during oxygen and nitrate respiration in Pseudomonas fluorescens}; Naumova RP et al.; A study of the metabolic pathway and the rate of 2,4,6-trinitrotoluene (TNT) transformation depending on the nature of the electron acceptor in the electron transport chain of Pseudomonas fluorescens B-3468 revealed that the first reaction of nitroreduction of TNT resulting in formation of 2-amino-4,6-dinitrotoluene (2A) and 4-amino-2,6-dinitrotoluene (4A) became more active in case of nitrate respiration as compared to oxygen respiration; a TNT decrease was 100 and 66%, respectively . The same tendency but much more pronounced was observed at the next stage of nitroreduction that lead to 2,4-diamino-6-nitrotoluene (2,4DA) . On the contrary, aerobic conditions are more preferable for the subsequent destruction of 2,4DA . Thus monoamino derivatives, 2A and 4A, predominated under anaerobic conditions, whereas 2,4DA under anaerobic ones (85 and 69% of the total nitrogen-containing metabolites), respectively . Phloroglucinol and pyrogallol accumulated in the culture liquid when the bacteria were grown on a medium containing 2,4DA as a sole source of nitrogen . Their role as intermediates was proved by the results obtained by studying oxidative activity of the cells grown in the presence of 2,4DA and phloroglucinol.

Br J Plast Surg, 1988 Jul, 41(4), 408 - 9
Cialit preserved cartilage: failure to guarantee sterility; Dickson WA et al.; Homograft cartilage preserved in Cialit solution became contaminated with Pseudomonas fluorescens, making it unsuitable for reconstructive surgery . Cialit solution has no virucidal activity, and the risk of viral infections such as AIDS and hepatitis is a further reason not to use it as a storage solution for cartilage.

Mol Gen Genet, 1988 Jul, 213(1), 112 - 7
The nucleotide sequence of a carboxymethylcellulase gene from Pseudomonas fluorescens subsp . cellulosa; Hall J et al.; The complete nucleotide sequence of the gene coding for one of the carboxymethylcellulases (CMCase), expressed by Pseudomonas fluorescens subsp . cellulosa, has been determined . The structural gene consists of an open reading frame, commencing with an ATG start codon, of 2886 base pairs followed by a TAA stop codon . The gene was shown to code for a signal peptide which closely resembles the signal peptides of other secreted proteins . Unlike most Pseudomonas genes, the CMCase sequence does not have a high G + C (51%) content and there is no marked preference for codons ending in G or C . Upstream of the structural gene there are no sequences which bear a strong resemblance to consensus Escherichia coli promoters . A sequence is present, however, which exhibits homology to the consensus DNA sequence that binds the catabolic activator protein (CAP) . Bal31 deletions of the structural gene revealed the extent by which the gene could be modified and still encode a functional CMCase . Subclones of the cellulase gene have been constructed in pUC18 and pUC19 . One of the resultant plasmids, pJHS1 directs a 20-fold increase in CMCase synthesis, when compared to the original construct, pJHH2 . Analysis of cells harbouring pJHS1 showed the cellulase polypeptide to have a molecular weight of 106000 . This is in close agreement with the predicted size of the enzyme deduced from the nucleotide sequence data.

Arch Biol Med Exp (Santiago), 1988 Jun, 21(1), 247 - 55
Biochemical and genetic studies of bacteria metabolizing lignin-related compounds; Vicuna R et al.; The ability of bacterial strains to metabolize lignin model compounds was studied . Strains examined were non-filamentous bacterial isolates obtained from decaying wood and the actinomycete Streptomyces viridosporus T7A . Model compounds included dimers containing either the beta-1 (1,2-diarylethane) or the beta-O-4 (arylglycerol-beta-aryl ether) type of linkage . Pseudomonas fluorescens biovar I A1 proliferated on anisoin (4,4'-dimethoxybenzoin) accumulating anisic acid temporarily . Cleavage at the beta-1 bond was also observed with crude extracts prepared from the same strain . In turn, cleavage of the beta-O-4 linkage of veratrylglycerol-beta-guaiacyl ether was detected in cultures of Pseudomonas acidovorans D3 . In this case, main degradation intermediates were beta-hydroxypropioveratrone, acetoveratrone and guaiacol . S . viridosporus T7A reduced the carbonyl group of some beta-1 dimers and did not modify the beta-O-4 model compounds tested . Attempts to ascribe a catabolic character to large molecular weight extrachromosomal DNA present in some strains were unsuccessful . Gene banks of P . fluorescens biovar I A1 and P . acidovorans D3 were prepared utilizing the broad host range cosmid pLAFR1 as vector.

J Dairy Sci, 1988 Jun, 71(6), 1432 - 8
Characterization of lipase of Pseudomonas fluorescens 27 based on fatty acid profiles; Ren TJ et al.; The objectives of this research were to isolate lipase from Pseudomonas fluorescens 27, to compare the purity of the partially purified lipase preparation with crude extract, and to determine if bands of lipase activity revealed by disc gel electrophoresis liberated different free fatty acids from milk fat . Lipases were isolated from a shaken skim milk culture of P . fluorescens 27 by using ion-exchange chromatography on DEAE cellulose (Whatman DE 32) and gel filtration on Sephadex G-150 . The principal lipase-rich fractions from gel filtration represented 6.2% of total lipolytic activity . Disc gel electrophoresis of partially purified enzyme revealed two protein bands . These protein bands were cut from disc electrophoresis gels and used as an enzyme source for reaction with butter oil . Free fatty acids were isolated from the assay mixture, separated, and quantified by gas chromatography . Data from gas chromatographic analysis indicated that P . fluorescens 27 produces at least two different lipases.

J Appl Bacteriol, 1988 May, 64(5), 403 - 7
Growth of psychrotrophic bacteria in raw and UHT-treated goats' milk; Cox JM et al.; The growth of six strains of Pseudomonas fluorescens, two of Ps . fragi, and one of Serratia liquefaciens was followed in raw and UHT-treated goats' milk, held at 4 degrees C . Generation times for Ps . fluorescens in UHT milk ranged from 5.19 to 5.81 h, increasing markedly in raw milk (8.34-21.49 h) . Growth of Ps . fragi did not differ significantly between raw (4.56, 4.65 h) and UHT (5.04, 7.24 h) milk . Generation times for S . liquefaciens were 6.63 and 14.07 h, for UHT and raw milk respectively.

J Antibiot (Tokyo), 1988 May, 41(5), 609 - 13
Antimycoplasmal activities of the pseudomonic acids and structure-activity relationships of monic acid A derivatives; Banks RM et al.; The antimycoplasmal activities of the pseudomonic acids isolated from Pseudomonas fluorescens NCIB 10586 are reported . Structure-activity relationships of a variety of ester, amide and thiol ester derivatives of the nucleus, monic acid A, are described . Enhanced antimycoplasmal activity is reported for a number of monic acid A esters and the most potent derivative, m-nitrobenzyl monate A, is a 100-fold more active against Mycoplasma hyopneumoniae than pseudomonic acid A.

J Bacteriol, 1988 May, 170(5), 2027 - 30
Attachment of Pseudomonas fluorescens to glass and influence of electrolytes on bacterium-substratum separation distance; Fletcher M; The influence of Na+, Ca2+, La3+, and Fe3+ on the adhesion of Pseudomonas fluorescens H2 and H2S was investigated with interference reflection microscopy (IRM) . IRM is a light microscopy technique which allows (i) visualization of the adhesive sites of living bacteria as they attach to a glass cover slip surface and (ii) evaluation of the bacterium-glass surface separation distance within a range of 0 to ca . 100 nm . The addition of each cation caused changes in IRM images consistent with a decrease in the separation distance, and minimum effective concentrations were as follows: Na+, 1 mM; Ca2+, 1 mM; La3+, 50 microM; and Fe3+, 50 microM . With strain H2, the effects of Na+, Ca2+, and La3+ were fully reversible in that the separation distance increased again when the electrolyte was replaced with distilled water . However, with strain H2S, a spontaneous mutant of H2 with increased attachment ability, only the effect of Na+ was fully reversible, and the effects of Ca2+ and La3+ were only partially reversible or irreversible . The effect of Fe3+ was irreversible with both strains, but this may be related not only to the electrolytic nature of Fe3+ but also to the decrease in solution pH to 3.5 caused by its addition . It is proposed that the electrolytes caused a decrease in separation distance by neutralizing negative charges on bacterial surface polymers and that the different effects obtained with the two strains are related to their different adhesion abilities.

J Clin Microbiol, 1988 May, 26(5), 1016 - 23
Common epitope on the lipopolysaccharide of Legionella pneumophila recognized by a monoclonal antibody; Barthe C et al.; Serogroup-specificity of Legionella pneumophila is related to lipopolysaccharide (LPS), and few cross-reactions between serogroups have been observed with rabbit or monkey antisera . C57BL/6 mice were sequentially immunized with crude outer membrane fractions of L . pneumophila serogroups 1, 5, and 7, Legionella bozemanii, and Legionella micdadei . Spleen cells from these mice were then fused with the Sp2-0/Ag14 mouse myeloma cell line . Outer membrane-rich fractions and LPS were prepared from L . pneumophila serogroups 1 to 8 and other Legionella and non-Legionella species . Immunoblots of these extracts were performed with monoclonal antibody obtained from these fusions . One of these monoclonal antibodies recognized an epitope common to all tested serogroups of L . pneumophila and attached to the major constituent of the outer membrane, LPS . This antibody did not react with other Legionella species and numerous gram-negative rods other than Pseudomonas fluorescens CDC93 . This monoclonal antibody may be useful in preliminary identification of L . pneumophila as an alternative to direct fluorescent-antibody testing.

Biochim Biophys Acta, 1988 Apr 14, 953(3), 258 - 62
Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens; Williamson G et al.; The oxidation-reduction potential of p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry . The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration . The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV . This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H . and Muller, F . (1987) Eur . J . Biochem . 167, 35-46) . Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV . No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed . These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme . These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.

Mikrobiologiia, 1988 Mar-Apr, 57(2), 218 - 22
{Possibilities for the deep bacterial destruction of 2,4,6-trinitrotoluene}; Naumova RP et al.; The possibility of 2,4,6-trinitrotoluene (TNT) deep destruction (the aromatic cycle fission inclusive) by Pseudomonas fluorescens B-3468 is reported for the first time . The formation of nitrogen-free metabolites, viz . phloroglucinol and pyrogallol, is preceded by the NAD(P)H-dependent deamination of 2,4-diamino-6-nitrotoluene (2,4-DA), a TNT intermediate . 30% of 2,4-DA nitrogen in released as ammonium under the action of an induced cell-free extract in the presence of the preferential co-factor NADH (together with FAD) . The elevated pyrogallol-decomposing activity in cells grown on 2,4-DA, phloroglucinol and pyrogallol as well as the induction of the pyrocatechase activity in cells grown on the above substrates, together with the earlier reported accumulation of phloroglucinol and pyrogallol upon 2,4-DA utilization, indicated that the enzyme might be involved in the TNT cycle cleavage . The participation of NADH-dependent glutamate dehydrogenase in 2,4-DA nitrogen utilization is supported by experimental evidence.

Biochem J, 1988 Mar 1, 250(2), 447 - 51
Pantothenases from pseudomonads produce either pantoyl lactone or pantoic acid; Airas RK; A pseudomonad was separated having a pantothenase that produced beta-alanine and pantoic acid . The pantothenase from an old strain, Pseudomonas fluorescens UK-1, was shown to produce beta-alanine and pantoyl lactone . The pantoic acid-producing pantothenase was characterized and compared with the pantothenase from the strain UK-1 . The Mr was 240,000; it apparently consists of four subunits . The Km value for pantothenate is 3 mM . The enzymic activity is affected by an ionizable group of pK 8.4, the enzyme is active at higher pH, and V but not Km is affected by pH . This pantothenase is not inhibited by di-isopropyl phosphorofluoridate or phenylmethanesulphonyl fluoride, unlike the enzyme from the strain UK-1 . Both pantothenases are inhibited by m-aminophenylboronic acid, oxalate, oxaloacetate and Cl- ions . The pantoic acid-producing pantothenase is inhibited also by SO4(2-) ions . The strong inhibitions by many compounds make this pantothenase unsuitable for the assay of pantothenic acid.

Int J Food Microbiol, 1988 Feb, 6(1), 51 - 6
Effect of carbon dioxide on growth and extracellular enzyme production by Pseudomonas fluorescens B52; Rowe MT; The effect of carbon dioxide (30 mM.l-1) on growth and extracellular protease and lipase production by Pseudomonas fluorescens B52 growing in a simulated milk medium at 7 degrees C in fermenter was investigated . The addition of carbon dioxide was shown to have a differential effect with extracellular enzyme production, particularly lipase, being inhibited to a greater extent than bacterial growth and final cell numbers.

Cell, 1988 Jan 15, 52(1), 19 - 26
The secondary structure of ribonuclease P RNA, the catalytic element of a ribonucleoprotein enzyme; James BD et al.; Secondary structure models for the ribonuclease (RNAase) P RNAs of Bacillus subtilis and E . coli were derived by a phylogenetic comparative analysis of published sequences as well as four novel ones . The RNAase P RNA genes from Bacillus megaterium, Bacillus brevis, Bacillus stearothermophilus, and Pseudomonas fluorescens were cloned, sequenced, and compared with the other available sequences . Regions of pairing were identified by the occurrence of homologous complementary sequences that vary among the compared molecules . A common core of primary and secondary structure can be identified in all these RNAase P RNAs . The previously noted striking differences between the Bacillus and the enteric RNAase P RNAs arise not only from point mutations, but from the addition or deletion of structural domains . The primary and secondary structural features that are common to all of the RNAase P RNAs are likely to be the elements involved in the binding and cleavage of tRNA precursors, and in the interaction with the RNAase P protein.

J Dairy Sci, 1988 Jan, 71(1), 41 - 5
Inhibition of lipolytic activity in milk by polysaccharides; Stern KK et al.; The effect of gums on the activity of milk lipase and a Pseudomonas lipase in milk was investigated . Gums were hydrated in water and mixed with whole milk . Lipase was added to the gum-milk mixture and hydrolysis was determined after 48 h at 4 degrees C by the acid degree value method . Of the gums tested, the anionically charged lambda-, t- and kappa-carrageenan, furcellaran, and sodium alginate significantly inhibited milk lipase activity by 93.7, 81.2, 46.8, 50.6, and 62.1%, respectively . Furthermore, lambda-carrageenan was 87.6% effective in inhibiting lipolysis by a purified Pseudomonas fluorescens MC50 lipase in milk . The other gums tested, tragacanth, carboxymethyl cellulose, locust bean, propylene glycol alginate, xanthan, microcrystalline cellulose, guar, and arabic did not significantly inhibit milk lipase . Commonly used stabilizers can inhibit lipolytic activity in milk.

Microbios, 1988, 56(226), 27 - 36
Metabolism of pyrimidine bases and nucleosides by Pseudomonas fluorescens biotype F; West TP; Pyrimidine metabolism in Pseudomonas fluorescens biotype F, and its ability to grow in liquid culture on pyrimidines and related compounds was investigated . It was found that uracil, uridine, cytosine, cytidine, deoxycytidine, dihydrouracil, dihydrothymine, beta-alanine or beta-aminoisobutyric acid could be utilized by this pseudomonad as a sole nitrogen source . Only uridine, cytidine, beta-alanine, beta-aminoisobutyric acid or ribose were capable of supporting its growth as a sole source of carbon . In solid medium, the pyrimidine analogue 5-fluorouracil or 5-fluorouridine could prevent P . fluorescens biotype F growth at a low concentration while a 20-fold higher concentration of 5-fluorocytosine, 5-fluorodeoxyuridine or 6-azauracil was necessary to block its growth . The pyrimidine salvage enzymes cytosine deaminase, nucleoside hydrolase, uridine phosphorylase, thymidine phosphorylase and cytidine deaminase were assayed . Only cytosine deaminase and nucleoside hydrolase activities could be detected under the assay conditions used . The effect of growth conditions on cytosine deaminase and nucleoside hydrolase levels in the micro-organism was explored . Cytosine deaminase activity was shown to increase if glycerol was substituted for glucose as the sole carbon source or if asparagine replaced (NH4)2SO4 as the sole nitrogen source in each respective medium . In contrast, nucleoside hydrolase activity remained virtually unchanged whether the carbon source in the medium was glucose or glycerol . A decrease in nucleoside hydrolase activity was witnessed when asparagine was present in the medium instead of (NH4)2SO4 as the sole source of nitrogen.

Arch Microbiol, 1988, 150(6), 523 - 8
Characterization of a pyoverdine-deficient mutant of Pseudomonas fluorescens impaired in the secretion of extracellular lipase; Fernandez L et al.; A mutant of Pseudomonas fluorescens strain B52 deficient in the synthesis of the fluorescent pigment, pyoverdine, was isolated . Absence of pyoverdine and other siderophores was confirmed by gel filtration, a specific siderophore assay, and inhibition studies with the iron chelator EDDA . Both parent and mutant synthesized additional outer membrane proteins in response to iron-limitation . Mutant cells cultured in the absence of iron(III) accumulated 55Fe-labeled pyoverdine . The mutant produced extracellular proteinase normally on various media, but was deficient in lipase secretion . Growth of the mutant with partially-purified pyoverdine resulted in a 2.5-fold stimulation of lipase secretion . The mutant grew poorly in deferrated medium; however, the addition of iron(III) stimulated growth . Proteinase secretion in deferrated medium was stimulated over a narrow range of iron(III) concentration, while lipase secretion was only slightly affected . The data suggest that separate regulatory mechanisms exist for the control of proteinase and lipase secretion by iron(III).

Vox Sang, 1988, 54(4), 201 - 4
A fatal transfusion reaction associated with blood contaminated with Pseudomonas fluorescens; Scott J et al.; A fatal transfusion reaction due to contamination of platelet-depleted whole blood with Pseudomonas fluorescens is reported . Routine sterility testing on blood products and environmental microbiological monitoring suggested no source for the contaminating organism, as has been the case for the majority of reported incidents of this type . The value of routine sterility testing in the prevention and investigation of such incidents is discussed.

Arch Microbiol, 1988, 149(5), 389 - 94
Degradation of diarylethane structures by Pseudomonas fluorescens biovar I; Gonzalez B et al.; Pseudomonas fluorescens biovar I was isolated from a pulp mill effluent based on its ability to grow on synthetic media containing 1,2-diarylethane structures as the sole carbon and energy source . Analysis of samples taken from cultures of this strain in benzoin or 4,4'-dimethoxybenzoin (anisoin), showed that cleavage between the two aliphatic carbons takes place prior to ring fission . Intermonomeric cleavage was also obtained with crude extracts . Substrates of this reaction were only those 1,2-diarylethane compounds that supported growth of the bacterium . The purification and partial characterization of an enzyme that catalyzes the NADH-dependent reduction of the carbonyl group of benzoin and anisoin is also reported.

J Bacteriol, 1988 Jan, 170(1), 380 - 5
Genetic determinants for catabolite induction of antibiotic biosynthesis in Pseudomonas fluorescens HV37a; Gutterson N et al.; Antibiotic biosynthesis is regulated by glucose in Pseudomonas fluorescens HV37a . Fusions between antibiotic biosynthetic operons (afu operons) and the Escherichia coli lac operon were isolated to evaluate the genetic determinants for the regulation of antibiotic biosynthesis . Four afu transcriptional units were defined, afuE, afuR, afuAB, and afuP . The afuE and afuR transcripts were promoted divergently at one locus and were catabolite induced, by 250-fold and 5-fold, respectively; the afuAB and afuP transcriptional units were not linked to the others and were not catabolite induced . Thus, regulation of afuE and afuR operon transcription is apparently the mechanism whereby glucose regulates antibiotic biosynthesis . Catabolite induction of the afuE and afuR transcriptional unit was dependent on the products of the afuA, afuB, and afuP genes . Expression of the afuE transcriptional unit was altered quantitatively in afuE mutants . Apparently the afuE transcriptional unit is regulated, at least in part, by its own gene products . Under inducing conditions, expression of the afuE, afuR, and afuP transcriptional units increased rapidly during a 6-h period.

Ciba Found Symp, 1988, 140, 3 - 15
Cyanide utilization and degradation by microorganisms; Knowles CJ; Various microorganisms can produce (cyanogenesis) or degrade cyanide . They degrade cyanide either to detoxify it, or to use it as a source of nitrogen for growth . Significant amounts of cyanide are formed as a secondary metabolite by a wide range of fungi and a few bacteria by decarboxylation of glycine . When cyanide has been formed by the snow mould fungus it is degraded by conversion to carbon dioxide and ammonia via an unknown pathway . In contrast, cyanogenic bacteria either do not further catabolize cyanide or they convert it into beta-cyanoalanine by addition to cysteine or O-acetylserine . Several non-cyanogenic fungi that are pathogens of cyanogenic plants are known to degrade cyanide by hydration to formamide by the enzyme cyanide hydratase . Such fungi can be immobilized and used in packed-cell columns to continuously detoxify cyanide . ICI Biological Products Business market a preparation of spray-dried fungal mycelia, 'CYCLEAR', to detoxify industrial wastes . Novo Industri have also introduced a cyanidase preparation to convert cyanide directly into formate and ammonia . Bacteria have been isolated that use cyanide as a source of nitrogen for growth . Because cyanide, as KCN or NaCN, is toxic for growth, the bacteria (Pseudomonas fluorescens) have to be grown in fed-batch culture with cyanide as the limiting nutrient . Cyanide is converted to carbon dioxide and ammonia (which is then assimilated) by an NADH-linked cyanide oxygenase system.

Mol Gen Genet, 1987 Dec, 210(3), 551 - 6
Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp . cellulosa; Gilbert HJ et al.; A genomic library of Pseudomonas fluorescens subsp . cellulosa DNA was constructed in bacteriophage lambda 47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated . A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively . Cells of Escherichia coli harbouring these plasmids expressed CMCase activity . The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis . pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter . All four cloned enzymes cleaved p-nitrophenyl-beta-D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity . In common with other extracellular enzymes cloned in E . coli, all the CMCases were exported to the periplasmic space in the enteric bacterium . The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E . coli.

Antimicrob Agents Chemother, 1987 Dec, 31(12), 1967 - 71
Revised structure for the phenazine antibiotic from Pseudomonas fluorescens 2-79 (NRRL B-15132); Brisbane PG et al.; A phenazine antibiotic (mp, 243 to 244 degrees C), isolated in a yield of 134 micrograms/ml from cultures of Pseudomonas fluorescens 2-79 (NRRL B-15132), was indistinguishable in all of its measured physicochemical (melting point, UV and infrared spectra, and gas chromatography-mass spectrometry data) and biological properties from synthetic phenazine-1-carboxylic acid . Gurusiddaiah et al . (S . Gurusiddaiah, D . M . Weller, A . Sarkar, and R . J . Cook, Antimicrob . Agents Chemother . 29:488-495, 1986) attributed a dimeric phenazine structure to an antibiotic with demonstrably similar properties obtained from the same bacterial strain . Direct comparison of the physicochemical properties of the authentic antibiotic obtained from D . M . Weller with synthetic phenazine-1-carboxylic acid and with the natural product from the present study established that all three samples were indistinguishable within the experimental error of each method . No evidence to support the existence of a biologically active dimeric species was obtained . Phenazine-1-carboxylic acid has a pKa of 4.24 +/- 0.01 (25 degrees C; I = 0.09), and its carboxylate anion shows no detectable antimicrobial activity compared with the active uncharged carboxylic acid species . These data suggest that phenazine-1-carboxylic acid is probably not an effective biological control agent for phytopathogens in environments with a pH greater than 7.

Can J Microbiol, 1987 Dec, 33(12), 1080 - 90
Purification and characterization of adhesive exopolysaccharides from Pseudomonas putida and Pseudomonas fluorescens; Read RR et al.; In this study, the adhesive exopolysaccharides of strains of Pseudomonas putida and P . fluorescens, both isolated from freshwater epilithic communities, were examined with regard to their chemical composition, biosynthesis, and their role in adhesion . Electron microscopy showed that both strains were enrobed in fibrous glycocalyces and that these structures were involved in attachment of the cells to a solid surface and as structural matrices in the microcolony mode of growth . In batch culture experiments most of the extracellular polysaccharide of both strains was found to be soluble in the growth medium rather than being associated with bacterial cells . Exopolysaccharide was synthesized during all phases of growth, but when growth was limited by exhaustion of the carbon source, exopolysaccharide synthesis ceased whereas exopolysaccharide synthesis continued for some time after cessation of growth in nitrogen-limited cultures . Exopolysaccharide from both strains was isolated and purified . Pseudomonas putida synthesized an exopolysaccharide composed of glucose, galactose, and pyruvate in a ratio of 1:1:1; the P . fluorescens polymer contained glucose, galactose, and pyruvate in a ratio of 1:1:0.5, respectively . Polymers from both strains were acetylated to a variable degree.

Mikrobiologiia, 1987 Nov-Dec, 56(6), 1033 - 4
{Nitro esterase activity of Pseudomonas fluorescens}; Il'inskaia ON et al.; The authors have demonstrated the biological nature of a process in which nitroester bonds are hydrolysed in the molecule of nitrocellulose . Pseudomonas fluorescens, strain 1/2, was isolated from a natural cenosis of microorganisms inhabiting active ooze, which was selected in a device for continuous cultivation by immobilisation on nitrocellulose fibers . The strain cleaves some nitro groups off the surface of nitrocellulose and the resultant nitrate ions are used in the process of assimilative reduction.

J Clin Microbiol, 1987 Nov, 25(11), 2080 - 4
Purified 60-kilodalton Legionella protein antigen with Legionella-specific and nonspecific epitopes; Plikaytis BB et al.; In a previous study, all convalescent-phase sera from patients with culture-confirmed legionellosis reacted on immunoblots with a Legionella genus-wide 58-kilodalton (kDa) protein antigen (J.S . Sampson, B.B . Plikaytis, and H.W . Wilkinson, J . Clin . Microbiol . 23:92-99, 1986) . The present study was done to immunologically characterize and determine the diagnostic relevance of this purified antigen . The antigen was precipitated from enriched cell extracts with ammonium sulfate and purified by high-pressure liquid chromatography . High-titered rabbit antiserum produced to the purified protein was used to show its presence on immunoblots in the 60-kDa range in 38 Legionella serogroups, representing 23 species, and in 39 non-Legionella bacteria . The antiserum was made specific for Legionella strains by sequential absorptions with Bordetella pertussis, Pseudomonas aeruginosa, and Pseudomonas fluorescens whole cells . Serum from legionellosis patients reacted with both specific and nonspecific epitopes . Results of indirect immunofluorescence experiments showed that neither specific nor nonspecific epitopes of the 60-kDa protein were surface exposed on Legionella cells and that cross-reactive epitopes were variably exposed on non-Legionella bacteria . The 60-kDa protein antigen should be useful in diagnostic tests for legionellosis if care is taken to expose cryptic epitopes and if the tests use or measure only the Legionella-specific epitopes.

Bioorg Khim, 1987 Oct, 13(10), 1428 - 9
{3-(3-hydroxy-2,3-dimethyl-5-oxoprolyl)amino-3,6-dideoxy-D-glucose: a new amino sugar of the antigenic polysaccharide from Pseudomonas fluorescens}; Naberezhnykh GA et al.; O-specific polysaccharide chain of the Pseudomonas fluorescens lipopolysaccharide is composed of 6-deoxy-L-talose, N-acetyl-D-fucosamine and N-acyl-3,6-dideoxy-D-glucose . Analysis of the latter sugar, obtained from the polysaccharide hydrolysate, by 1H NMR (including NOE), 13C NMR, and FAB mass spectrometry proved the unusual N-acyl substituent to be a 3-hydroxy-2,3-dimethyl-5-oxoproline residue.

Eur J Biochem, 1987 Aug 17, 167(1), 35 - 46
The elucidation of the microheterogeneity of highly purified p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by various biochemical techniques; Van Berkel WJ et al.; Highly purified p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens can be separated into at least five fractions by anion-exchange chromatography . All fractions exhibit the same specific activity and the enzyme exists mainly in the dimeric form in solution . Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of a mixture of the different fractions reveals two apparent forms of enzyme molecules, while isoelectric focusing experiments, on the other hand, reveal six apparently different forms of enzyme molecules . It is shown that the different forms of enzyme molecules are due to the (partial) oxidation of Cys-116 in the sequence of the enzyme . This interpretation of the data is supported by kinetic measurements of the formation of hybrid dimeric molecules monitored by fast protein liquid chromatography, using purified enzyme containing Cys-116 either in the native and or the fully oxidized (sulfonic acid) state . By chemical modification studies using maleimide derivatives, 5,5'-dithiobis(2-nitrobenzoate) and H2O2, it is shown that sulfenic, sulfinic and sulfonic acid derivatives of Cys-116 are products of oxidation . The results are briefly discussed with respect to the possibility that this isolation artifact might also be partially responsible for the appearance of multiple forms of enzyme molecules in other biochemical preparations.

J Bacteriol, 1987 Aug, 169(8), 3853 - 6
Decreased chromate uptake in Pseudomonas fluorescens carrying a chromate resistance plasmid; Ohtake H et al.; CrO4(2-) resistance in Pseudomonas fluorescens LB300(pLHB1) was related to reduced uptake of CrO4(2-) relative to the plasmidless strain LB303 . 51CrO4(2-) was transported mainly via the SO4(2-) active transport system; thus, cells grown with 0.15 mM cysteine, a repressor of the SO4(2-) transport system, were much more resistant to CrO4(2-) than those grown with 0.15 mM djenkolic acid, which derepressed the 35SrO4(2-) uptake system . Kinetics of 51CrO4(2-) uptake by P . fluorescens with and without the plasmid showed that the Vmax for 51CrO4(2-) uptake with the resistant strain was 2.2 times less than the Vmax for the sensitive strain, whereas the Km remained constant.

Appl Environ Microbiol, 1987 Aug, 53(8), 1973 - 6
Effect of temperature shifts on extracellular proteinase-specific mRNA pools in Pseudomonas fluorescens B52; McKellar RC et al.; The influence of a shift in temperature from 20 to 32 degrees C on extracellular proteinase synthesis by Pseudomonas fluorescens B52 was examined . When cells actively synthesizing proteinase at 20 degrees C were shifted to 32 degrees C, enzyme synthesis ceased immediately . After 30 min at 32 degrees C, cells recovered at 20 degrees C after a lag of 30 min . Rifampin and chloramphenicol prevented recovery of synthesis at 20 degrees C . Rifampin-insensitive proteinase synthesis (an indirect measure of proteinase-specific mRNA pools) decreased after the exposure of cells to 32 degrees C for 30 min but was recovered during incubation at 20 degrees C . Controls not exposed to a temperature shift experienced no loss of rifampin-independent synthesis . Cells experienced a 50% reduction in mRNA pools after 15 min at 32 degrees C . The data support the working hypothesis that the loss of mRNA pools after treatment at 32 degrees C is responsible for the lag before the recovery of extracellular proteinase synthesis.

Mikrobiologiia, 1987 Jul-Aug, 56(4), 635 - 41
{Kinetics of Pseudomonas fluorescens growth under different conditions of culturing}; Aminov RI et al.; The dynamics of Pseudomonas fluorescens VKM-1472 growth was studied under the conditions of batch and continuous cultivation, and the behaviour of the organism was investigated upon nutrition shifts and starvation . At D greater than 0.375 h-1, just as in the batch culture with a substrate excess, the strain realised excessive metabolism {15}: the yield in terms of the substrate fell down (38% for the batch culture and 40% for the continuous culture), the titre of viable cells decreased to a considerable extent, and maintenance expenditures increased (3 times for qgluc and 7.5 times for qO2) . When the culture was incubated under the oligotrophous conditions, the biomass yield decreased fourfold within 8 days and the titre of viable cells dropped down twofold within the same period of time . However, the organism was still capable of oxidising a wide range of substrates (17 substrates were studied) . As soon as a substrate was added, it was oxidised at a high rate and changes in the macromolecular composition were characteristic of an "up-jump" in the growth rate {11} . It is possible that the growth and behaviour of this organism are associated with its ecological niche occupied in natural systems.

J Bacteriol, 1987 Jun, 169(6), 2769 - 73
Flagella of a plant-growth-stimulating Pseudomonas fluorescens strain are required for colonization of potato roots; De Weger LA et al.; The role of motility in the colonization of potato roots by Pseudomonas bacteria was studied . Four Tn5-induced flagella-less mutants of the plant-growth-stimulating P . fluorescens WCS374 appeared to be impaired in their ability to colonize growing potato roots.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1987 Jun, 265(1-2), 62 - 73
{Automated micromethod for the determination of the utilization of carbon sources by clinically significant Pseudomonas species}; Kampfer P et al.; The assimilation of 43 different carbon substrates by 93 clinical strains of Pseudomonas aeruginosa was studied by a new miniaturized rapid method . Reading of assimilation results was done photometrically after 18-20 h incubation and the resulting data were captured and stored by a microcomputer . The differentiating capacity of the assimilation tests were verified by comparing the results of 41 strains of Pseudomonas fluorescens, 48 strains of Pseudomonas putida, 52 strains of Pseudomonas maltophilia and respectively 10 strains of Pseudomonas pseudomallei and Pseudomonas cepacia . The assimilation pattern obtained from the Pseudomonas aeruginosa strains agreed to those described in literature and because of miniaturization, standardisation, facility of use and automatic reading the method seems to be suitable for routine laboratory work.

Biotechnol Appl Biochem, 1987 Jun, 9(3), 251 - 7
Studies on the enzymatic hydrolysis of amino acid carbamates; Sambale C et al.; Several commercially available enzymes were tested for their ability to hydrolyze amino acid carbamates . No activity was found with pig liver esterase, the hydantoinase from Pseudomonas fluorescens DSM 84, or the urease from jack beans . A stereoselective cleavage of the carbamyl group yielding L-amino acids was observed by acylase and acetylcholinesterases from bovine and human erythrocytes . Racemic mixtures of N-(methoxycarbonyl)-DL-alanine, N-(ethoxycarbonyl)-DL-alanine, and the corresponding valine carbamates are hydrolyzed to L-alanine and L-valine, respectively, by acylases leaving the D-amino acid carbamates unchanged . The lysine carbamates were not hydrolyzed by acylases . In contrast only the methoxycarbonyl amino acids were split by acetylcholinesterases, which, however, also cleave alpha, epsilon-(N-methoxycarbonyl)-DL-lysine stereoselectively at the alpha position, yielding epsilon-N-methoxycarbonyl-L-lysine . The optimum pH for enzymatic activity of hog kidney acylase was 7.5 and a Km value of 8.2 mM for N-(methoxycarbonyl)-DL-alanine was determined . For the acetylcholinesterases the reaction rate reaches an optimum between pH 7.5 and 8 . The Km value was 68 mM for N-(methoxycarbonyl)-DL-alanine.

J Biol Chem, 1987 May 5, 262(13), 6060 - 8
para-Hydroxybenzoate hydroxylase containing 6-hydroxy-FAD is an effective enzyme with modified reaction mechanisms; Entsch B et al.; The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens, was replaced by 6-hydroxy-FAD (an extra hydroxyl group on the carbon at position 6 of the isoalloxazine ring of FAD) . The catalytic cycle of this modified enzyme was analyzed and compared to the function of native (FAD) enzyme . Transient state kinetic analyses of the multiple changes in the chemical state of the flavin were the principal methods used to probe the mechanism . Four known substrates of the native enzyme were used to probe the reaction . With the natural substrate, p-hydroxybenzoate, the 6-hydroxy-FAD enzyme activity was 12-15% of native enzyme, due to a slower release of product from the enzyme, and less than one product molecule was formed per NADPH oxidized, due to an increased rate of nonproductive decomposition of the transient peroxyflavin essential to the catalytic pathway . More extensive changes in mechanism were observed with the substrates, 2,4-dihydroxybenzoate and p-aminobenzoate . The results suggest that, during catalysis, when the reduced state of FAD is ready for oxygen reaction, the substrate is located below and close to the C-4a/N-5 edge of the isoalloxazine ring . The nature of the high extinction, transient state of flavin, formed upon transfer of oxygen to substrate is discussed . It is not a flavin cation, and is unlikely to be an oxygen-substituted analogue of N-3/C-4 dihydroflavin.

J Dairy Res, 1987 May, 54(2), 295 - 302
Assay of proteinases of psychrotrophic bacteria in milk using hide powder azure and a simple procedure for clarification of milk; Stead D; The clarification of milk by addition of solutions of Triton X-100 and EDTA after digestion of added Hide Powder Azure (HPA) was found to provide a simple method of determining the extracellular proteinase activity of Gram-negative psychrotrophic bacteria in pasteurized whole milk . The light absorbance of the clarified HPA digestion product was measured directly, after a brief incubation period, and was stable to storage of samples in diffuse daylight for at least 2 d . Proteinase produced by growth in refrigerated whole milk of as few as 2.5 X 10(6) cfu ml-1 of Pseudomonas fluorescens AR11 was detected.

J Dairy Res, 1987 May, 54(2), 283 - 93
Peptidases from two strains of Pseudomonas fluorescens: partial purification, properties and action on milk; Shamsuzzaman K et al.; Pseudomonas fluorescens strains 240 and 32A expressed cell-associated peptidase activity which was shown by subcellular fractionation to be primarily intracellular . Two peptidases were partly purified from strain 32A . One specifically hydrolysed N-alpha-benzoyl-DL-arginine-4-nitroanilide and was termed endopeptidase and the other hydrolysed L-lysine- and L-leucine-4-nitroanilide and was termed aminopeptidase . The endopeptidase had very low activity on bovine serum albumin compared with that of trypsin and probably was not a proteinase . The endopeptidase had a mol . wt of 33,000 and a pH optimum of 8.0 . The enzyme was stimulated by Ca2+ and Mg2+ and inhibited by Co2+, Mn2+, Hg2+, Zn2+ and leupeptin . Soya bean trypsin inhibitor and phenylmethane sulphonyl fluoride (PMSF) had no effect on its activity . The aminopeptidase had a mol . wt of 44,000 and a pH optimum of 8.0 . It was inhibited by all the metal ions mentioned above and by PMSF . Little proteolysis was found when ultra high temperature (UHT) sterilized milk was treated with cell-free extract from strain 32A . It was concluded that the cell-associated peptidases from Pseudomonas strains normally present in raw milk may not contribute significantly to the deterioration of UHT sterilized milk.

J Hosp Infect, 1987 May, 9(3), 243 - 8
Blood transfusion-associated Pseudomonas fluorescens septicaemia: is this an increasing problem?
Murray AE, Bartzokas CA, Shepherd AJ, Roberts FM.
Pseudomonas fluorescens transfusion-related septicaemia (TRS) is rare . We present the first description in the UK of two cases of TRS caused by this organism.

Arch Microbiol, 1987 Apr, 147(3), 225 - 30
Influence of iron(III) and pyoverdine on extracellular proteinase and lipase production by Pseudomonas fluorescens B52; McKellar RC et al.; Factors associated with the production of extracellular lipase and proteinase by Pseudomonas fluorescens B52 during the late-log, early-stationary phase of grown were examined . Active lipase production by resting cell suspensions was observed when cells were harvested during the log phase (A600 of 0.3-0.9) . Resting suspensions of younger cells (A6000 less than 0.1) synthesized lipase after a significant lag . Addition of cells of the proteinase- and lipase-deficient mutant P . fluorescens RM14 to B52 cells at low density resulted in stimulation of lipase and proteinase production . Similar results were found using cell-free culture fluid of RM14 . Gel filtration on Biogel P2 revealed that the stimulatory factor co-chromatographed with the iron(III) siderophore, pyoverdine . Partially purified pyoverdine stimulated enzyme synthesis at a concentration of 6 microM while having no effect on activity of preformed enzyme . Production of pyoverdine and extracellular enzymes was also stimulated by transferrin, a strong iron(III) binding protein . Growth of B52 in deferrated media was limited to 27% of that found with untreated media . Maximum pyoverdine, proteinase and lipase synthesis was obtained at a final iron(III) concentration of 5.75 microM . Growth was maximal in 8.75 microM iron(III) while synthesis of pyoverdine, proteinase and lipase was reduced to 3.6, 6.6 and 30% respectively in 23.75 microM iron(III) . Lipase activity in cell-free culture fluid was slightly inhibited by the addition of up to 400 microM iron(III) while proteinase activity was unaffected . In dilute cell suspensions, lipase synthesis was more sensitive to iron(III) than was proteinase (50% inhibition at 1.6 microM and a maximum of 40% inhibition at 5.0 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1987 Mar 16, 163(3), 535 - 44
Chemical modification of arginine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: a kinetic and fluorescence study; Wijnands RA et al.; The flavoprotein p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was modified by several arginine-specific reagents . Modifications by 2,3-butanedione led to the loss of activity of the enzyme, but the binding of p-hydroxybenzoate and NADPH to the enzyme was little or not at all affected . However the formation of the enzyme-substrate complex of the modified enzyme was accompanied by an increase of the fluorescence of protein-bound FAD, in contrast to that of native enzyme which leads to quenching of the fluorescence . Enzyme modified by phenylglyoxal did not bind p-hydroxybenzoate nor NADPH . Quantification and protection experiments showed that two arginine residues are essential and a model is described which accounts for the results . Modification by 4-hydroxy-3-nitrophenylglyoxal reduced the affinity of the enzyme for the substrate and NADPH . The ligand; Fairbairn DJ et al.; Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied . Proteinase production was optimal at 20 degrees C and pH 6.9 in static culture when calcium was included in the medium . Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds . The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids . The organism did not utilize lactose, the most abundant carbohydrate in milk . Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production . A medium containing sodium caseinate and pyruvate supported good growth and enzyme production . All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production . Asparagine was the most effective amino acid inducer . Particular combinations of amino acids could induce or repress proteinase production . The regulation of proteinase production by Ps . fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression . The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.

J Dairy Res, 1987 Feb, 54(1), 51 - 60
Mechanisms of heat inactivation of a proteinase from Pseudomonas fluorescens biotype I; Diermayr P et al.; Heat inactivation of a metalloproteinase, isolated from Pseudomonas fluorescens biotype I strain 112, was investigated in the temperature ranges 50-60 degrees C and 90-140 degrees C . At 90 degrees C the denaturation of the ; Fairbairn DJ et al.; Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied . Proteinase production was optimal at 20 degrees C and pH 6.9 in static culture when calcium was included in the medium . Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds . The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids . The organism did not utilize lactose, the most abundant carbohydrate in milk . Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production . A medium containing sodium caseinate and pyruvate supported good growth and enzyme production . All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production . Asparagine was the most effective amino acid inducer . Particular combinations of amino acids could induce or repress proteinase production . The regulation of proteinase production by Ps . fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression . The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.

J Dairy Res, 1987 Feb, 54(1), 51 - 60
Mechanisms of heat inactivation of a proteinase from Pseudomonas fluorescens biotype I; Diermayr P et al.; Heat inactivation of a metalloproteinase, isolated from Pseudomonas fluorescens biotype I strain 112, was investigated in the temperature ranges 50-60 degrees C and 90-140 degrees C . At 90 degrees C the denaturation of the enzyme followed first-order kinetics with a decimal reduction time of 110 min and a velocity constant K of 3.5 X 10(-4) S-1 . Activation energy Ea was 100 kJ/mol for this temperature range . In the 50-60 degrees C region the proteinase was inactivated by autolysis, as shown by electrophoresis and gel filtration . At 55 degrees C the decimal reduction time was approximately 22 s, at 57 degrees C it was 8 s . Rapid inactivation at 55 degrees C was only possible if the enzyme was heated from lower temperatures, but not if cooled down from 90 degrees C . This is due to a conformational change of the protein at this temperature . A model for the description of heat inactivation in the two temperature ranges is proposed.

J Appl Bacteriol, 1987 Feb, 62(2), 119 - 28
Water relations of solute accumulation in Pseudomonas fluorescens; Prior BA et al.; When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (aw) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool . This increase was not observed when the aw of the medium was reduced to 0.980 with sorbitol . Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium . In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 aw . The maintenance coefficients for glucose uptake were similar at both aw values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 aw.

J Enzyme Inhib, 1987, 1(3), 215 - 22
Inactivation of GABA aminotransferase by 3-nitro-1-propanamine; Alston TA et al.; 3-Nitro-1-propanamine is a close structural analog of the neuro-transmitter GABA . The nitro compound is a good substrate for the GABA aminotransferase from porcine brain . However, it inactivates the GABA aminotransferase from GABA-grown Pseudomonas fluorescens in a slowly reversible reaction . Both enzymes are inactivated by the homolog 4-nitro-1-butanamine.

Biol Chem Hoppe Seyler, 1987 Jan, 368(1), 57 - 61
Influence of EDTA and metal ions on a metalloproteinase from Pseudomonas fluorescens biotype I; Diermayr P et al.; The inactivation of a metalloproteinase from Pseudomonas fluorescens Biotype I with EDTA was investigated at 22 degrees C and 37 degrees C . At 22 degrees C proteolytic activity decreases linearly with time and an inactive apoenzyme is obtained by dialysis . Proteolytic activity can be restored with several metal-ions, Ca2+, Zn2+, Mg2+, Sr2+ and co2+ give the best results . Activity and substrate specificity are influenced by the metal-ions . Reactivation depends on the concentration of the metal-ions, optimum concentration is 1 mM for Ca2+ and 50 microM for Zn2+ . The isoelectric point of the apoenzyme is around 8.0, this is about 0.3 pH-units lower than the isoelectric point of the native proteinase . At 37 degrees C inactivation follows first order kinetics and is irreversible because of autolysis as shown by a gel filtration-experiment.

Gene, 1987, 51(1), 91 - 6
IS50L as a non-self transposable vector used to integrate the Bacillus thuringiensis delta-endotoxin gene into the chromosome of root-colonizing pseudomonads; Obukowicz MG et al.; Insertion sequence IS50L of transposon Tn5 was used as a non-self transposable vector to integrate the delta-endotoxin gene (tox) from Bacillus thuringiensis subsp . kurstaki HD-1 into the chromosome of two corn-root colonizing strains of Pseudomonas fluorescens (112-12 and Ps3732-3-7) . A DNA fragment containing the KmR gene from Tn5 and tox was inserted into an IS50L element (IS50L-tox) contained on a suicide plasmid . Transposition of IS50L-tox into the chromosome of P . fluorescens 112-12 and Ps3732-3-7 occurred by selecting for KmR transconjugants and supplying transposase in cis from a linked IS50R element . A frameshift mutation in the transposase gene of the IS50L-tox element was also constructed to decrease the likelihood that suppression or a spontaneous reversion at the UAA (ochre) termination codon of IS50L would create an active transposase . The inability of IS50L-tox to transpose further minimizes the potential for horizontal gene transfer of the tox gene to other bacterial species . Expression of the Tox protein in strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).

Gene, 1987, 61(3), 299 - 306
An efficient mobilizable cosmid vector, pRK7813, and its use in a rapid method for marker exchange in Pseudomonas fluorescens strain HV37a; Jones JD et al.; We describe the construction and utilization of a new mobilizable cosmid vector . Using this vector, mobilizable libraries of bacterial DNA can be efficiently made without a need for size fractionation of target DNA . The low stability of this vector in Pseudomonas fluorescens makes it useful in a rapid strategy, which is not dependent on plasmid incompatibility, for recombining transposon-induced mutations into the bacterial chromosome.

Can J Microbiol, 1987 Jan, 33(1), 63 - 9
Phosphate-selective porins from the outer membranes of fluorescent Pseudomonas sp; Poole K et al.; Phosphate starvation induced oligomeric proteins from the outer membranes of Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aureofaciens, and Pseudomonas chlororaphis were purified to homogeneity . The incorporation of the purified proteins into planar lipid bilayer membranes resulted in stepwise increases in membrane conductance . Single channel conductance experiments demonstrated that these proteins were all capable of forming small channels, similar to the Pseudomonas aeruginosa phospsate porin protein P, with average single channel conductances in 1 M KCl of between 233 and 252 pS . Single channel conductance measurements made in salts of varying cation or anion size indicated that the channels were uniformly anion selective . The measurement of single channel conductance as a function of KCl concentration revealed that all channels saturated at higher salt concentrations, consistent with the presence of an anion-binding site in the channel . Apparent Kd values for Cl- binding were calculated and shown to vary only twofold (180-297 mM) among all channels, including protein P channels . Phosphate competitively inhibited chloride conductance through these channels with apparent I50 values of between 0.59 and 2.5 mM phosphate at 40 mM Cl- and between 9.7 and 27 mM phosphate at 1 m Cl- . These data were consistent with the presence of a phosphate-binding site in the channels of these phosphate-regulated proteins . Furthermore, they indicated that these channels exhibit at least a 20- to 80-fold higher affinity for phosphate than for chloride.

J Biol Chem, 1986 Dec 15, 261(35), 16295 - 7
Evidence that two covalent intermediates, phosphoryl and malonyl enzymes, are formed during malonyl-coenzyme A synthetase catalysis; Kim YS et al.; The isolation of malonyl-coenzyme A synthetase from Pseudomonas fluorescens grown on malonate has been reported recently (Kim, Y.S., and Bang, S.K . (1985) J . Biol.Chem . 260, 5098-5104) . This enzyme is phosphorylated in the presence of ATP and Mg2+ . The phosphoryl group appears on one subunit of the enzyme composed of two different subunits, and the phosphoryl enzyme is acid labile and base stable . The phosphoryl group on the enzyme is released by the incubation of the phosphoryl enzyme with malonate and malonyl enzyme is formed . The malonyl enzyme is acid labile and also relatively unstable under basic conditions . The malonyl group is found on the subunit of the enzyme which is phosphorylated . Malonyl-CoA is formed when malonyl enzyme reacts with coenzyme A . These results suggest that two convalent intermediates, phosphoryl and malonyl enzyme, are sequentially formed in the synthesis of malonyl-coenzyme A by malonyl-coenzyme A synthetase catalysis.

Biotechnol Appl Biochem, 1986 Dec, 8(6), 564 - 74
Characterization of hydantoinase from Pseudomonas fluorescens strain DSM 84; Morin A et al.; The hydantoinase (EC 3.5.2.2) from Pseudomonas fluorescens strain DSM 84 was purified either by hydrophobic interaction chromatography on phenyl-Sepharose or by salting out chromatography on Sepharose 4B, gel filtration on Sephacryl S-400, and preparative electrophoresis . Molecular weight values of 230,000 and 60,000 for the native enzyme and each of the four subunits were estimated for the hydantoin hydrolysing activity . The hydantoinase was stable at temperatures up to 40 degrees C but showed an optimal activity at 55 degrees C . The enzyme was markedly inhibited by copper, para-hydroxymercuribenzoate, 8-hydroxyquinoline, and 2,2'-dipyridyl but not by zinc, and poorly by EDTA and o-phenanthroline . The hydantoin-hydrolyzing activity could be reactivated by ferrous ions . Dihydrouracil was the most readily hydrolyzed substrate . The dihydropyrimidinase produced by strain DSM 84 could also hydrolyze 5-substituted hydantions such as isopropylhydantoin (valine derivative) continuously for 10 days in a membrane reactor at a conversion rate of 30% . The only identified end product was N-carbamyl-D-valine.

Bioorg Khim, 1986 Dec, 12(12), 1641 - 8
{The structure of O-specific polysaccharide chain of Pseudomonas fluorescens lipopolysaccharide}; Khomenko VA et al.; An O-specific polysaccharide, containing 6-deoxy-L-talose (6dTal), N-acetyl-D-fucosamine (FucNAc), 3-amino-3,6-dideoxy-D-glucose with an unidentified N-acyl substituent (Qui3NR), and O-acetyl groups, was obtained on mild acid degradation of a Pseudomonas fluorescens strain 361 lipopolysaccharide . On the basis of O-deacetylation, acid hydrolysis, methylation, selective solvolysis with anhydrous hydrogen fluoride, and 13C NMR analysis, the polysaccharide is built up of trisaccharide repeating units of the following structure: (Formula: see text).

Mikrobiologiia, 1986 Nov-Dec, 55(6), 1040 - 1
{Final stages of the preliminary metabolism of 2,4,6-trinitrotoluene in Pseudomonas fluorescens}; Selizanovskaia SIu et al.; The work was aimed at studying the transformation of 2,4-diamino-6-nitrotoluene (2,4-DA), an intermediate product in 2,4,6-trinitrotoluene catabolism by microorganisms . The results allow one to propose the following scheme for the terminal steps of TNT preparatory metabolism: 2,4-DA----{phloroglucinol carboxylic acid}----phloroglucinol----pyrogallol----ring cleavage.

J Appl Bacteriol, 1986 Nov, 61(5), 395 - 400
Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk; Shelley AW et al.; The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads . Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5 degrees to 30 degrees C . Pseudomonas fluorescens was the most frequently encountered species but Ps . fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures . A representative strain of Ps . fragi multiplied faster in cold-stored milk than did three representative strains of Ps . fluorescens . The lipases produced by Ps . fragi strains were more heat-stable than those produced by Ps . fluorescens strains.

Appl Environ Microbiol, 1986 Nov, 52(5), 1190 - 4
DNA hybridization probe for the Pseudomonas fluorescens group; Festl H et al.; Plasmid pHF360 was constructed from cloned rRNA genes (rDNA) of Pseudomonas aeruginosa and used as hybridization probe for the Pseudomonas fluorescens group . The probe was tested by dot and in situ colony hybridizations to chromosomal DNAs from a wide variety of organisms . pHF360 DNA hybridized exclusively to chromosomal DNAs from bacteria representing the P . fluorescens group and separated them clearly from all other bacteria tested in the present study . Determination of the nucleotide sequence of the cloned DNA showed that it is a fragment from a 23S rRNA gene of P . aeruginosa . It was compared with the published 23S RNA sequence from Escherichia coli.

Appl Environ Microbiol, 1986 Nov, 52(5), 1183 - 9
Multiple antibiotics produced by Pseudomonas fluorescens HV37a and their differential regulation by glucose; James DW Jr et al.; Pseudomonas fluorescens HV37a inhibited growth of the fungus Pythium ultimum on potato dextrose agar (PDA) . An antibiotic activity produced under these conditions was fractionated and partially characterized . Extracts prepared from the PDA on which HV37a was grown revealed a single peak of antibiotic activity on thin-layer chromatograms . Similar extracts were prepared from mutants of HV37a . Their analysis indicated that the antibiotic observed in thin-layer chromatograms was responsible for fungal inhibition observed on PDA . The production of the PDA antibiotic required the presence of glucose, whereas two other antibiotic activities were produced only on potato agar without added glucose . Two mutants (denoted AfuIa and AfuIb) previously characterized as deficient in fungal inhibition on PDA showed altered regulation of the production of all three antibiotics in response to glucose . These mutants were also deficient in glucose dehydrogenase . Mutants isolated as deficient in glucose dehydrogenase were also deficient in fungal inhibition and were grouped into two classes on the basis of complementation analysis with an AfuI cosmid . Glucose regulation of antibiotic biosynthesis therefore involves at least two components and requires glucose dehydrogenase.

J Bacteriol, 1986 Nov, 168(2), 982 - 9
Tn5-mediated integration of the delta-endotoxin gene from Bacillus thuringiensis into the chromosome of root-colonizing pseudomonads; Obukowicz MG et al.; Gene replacement mediated by Tn5 sequences was used to integrate the Bacillus thuringiensis subsp . kurstaki HD-1 delta-endotoxin gene (tox) into the chromosome of two corn root-colonizing strains of Pseudomonas fluorescens . A Tn5 transposase deletion element containing the tox gene (delta Tn5-tox) was substituted for a Tn5 element previously present in the P . fluorescens chromosome . Two classes of delta Tn5-tox elements were made . The first class encodes kanamycin resistance in addition to the Tox protein, whereas the second class encodes only the Tox protein . Both classes of delta Tn5-tox elements can no longer transpose, owing to a 324-base-pair deletion in the transposase gene of IS50R, minimizing the potential for horizontal gene transfer of the tox gene to other bacterial species . A frameshift mutation in the transposase gene of IS50L was also constructed to eliminate the possibility of suppression or of a spontaneous reversion at the ochre termination codon that would create an active transposase . Expression of the Tox protein in P . fluorescens strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).

Nucleic Acids Res, 1986 Oct 24, 14(20), 8047 - 60
Conserved repeats in diverged ice nucleation structural genes from two species of Pseudomonas; Warren G et al.; Sequence analysis shows that an ice nucleation gene (inaW) from Pseudomonas fluorescens is related to the inaZ gene of Pseudomonas syringae . The two genes have diverged by many amino acid substitutions, and have effectively randomized the third bases of homologous codons . By reference to their potential for change, it is shown that certain conserved features must have been maintained by selection pressure . In particular, their conservation of internal sequence repetition, with three orders of repeat periodicity in each gene, suggests that the pattern of repetition is significant to the gene products' function . We propose models for the structure of the gene products in which each order of periodicity would be required for the nucleation function.

Mikrobiologiia, 1986 Sep-Oct, 55(5), 816 - 20
{Intracellular development of Bdellovibrio bacteriovorus}; Konovalova SM et al.; The intracellular growth of Bdellovibrio bacteriovorus, a bacterial parasite, was studied by a light-optical method using time-lapse cinemicrography . The organism was found to be capable of growth in the periplasmic space of filamentous cells of the host bacterium Pseudomonas fluorescens without any contact with the cytoplasmic membrane . Several B . bacteriovorus cells could grow simultaneously in the bdelloplasm.

J Biochem (Tokyo), 1986 Sep, 100(3), 697 - 705
Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens; Takagi JS et al.; The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2,066-base-pair DNA fragment containing the aspA gene was determined . The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein . The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al . (1984) J . Biochem . 96, 545-552) . These results indicate that aspartase of P . fluorescens consists of four identical subunits with a molecular weight of 50,859, composed of 472 amino acid residues . The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures . Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator . The G + C content of the coding sequence was found to be 62.3% . Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position . The deduced amino acid sequence was 56.3% homologous with that of the enzyme of E . coli W (Takagi et al . (1985) Nucl . Acids Res . 13, 2063-2074) . Cys-140 and Cys-430 of the E . coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J . Biochem . 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P . fluorescens enzyme.

J Dairy Res, 1986 Aug, 53(3), 457 - 66
Purification and characterization of the extracellular proteinase of Pseudomonas fluorescens NCDO 2085; Fairbairn DJ et al.; Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 degrees C and pH 7.0 . The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography . The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3.5% of the original activity . The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5.40 +/- 0.05 and a mol . wt of 40 200 +/- 2100 . It is heat-stable having D-values at 74 and 140 degrees C of 1.6 and 1.0 min respectively; 40 and 70% of the original activity remained after HTST (74 degrees C/17 s) and ultra high temperature (140 degrees C/4 s) treatments respectively . The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.

Biochem Biophys Res Commun, 1986 Jul 31, 138(2), 568 - 72
L-aspartate ammonia-lyase and fumarate hydratase share extensive sequence homology; Takagi JS et al.; Based on our recent determinations of the nucleotide sequences of the L-aspartate ammonia-lyase genes from Escherichia coli and Pseudomonas fluorescens, primary structures of the two L-aspartate ammonia-lyases and fumarate hydratases from Bacillus subtilis and E . coli (N-terminal partial sequence) were compared by computer analysis . These four enzymes exhibited a significant homology of at least 37%, implying that L-aspartate ammonia-lyase and fumarate hydratase share a common evolutionary origin . To authors' knowledge, this feature appears to be the first example showing that two kinds of enzymes catalyzing different types of reactions, albeit similar, share such a high degree of sequence homology.

Biochemistry, 1986 Jul 29, 25(15), 4211 - 8
Chemical modification of tyrosine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: assignment in sequence and catalytic involvement; Wijnands RA et al.; p-Hydroxybenzoate hydroxylase was modified by diethyl pyrocarbonate at pH values greater than 7 and by p-diazobenzoate . Modification of the enzyme by diethyl pyrocarbonate abolishes the affinity of the enzyme for the substrate p-hydroxybenzoate . Modification by p-diazobenzoate has the same effect on the enzyme . The enzyme is protected against these modifications by the effector p-fluorobenzoate . The data indicate that the modification of one tyrosine residue in the active center of the enzyme is responsible for the loss of enzyme activity . This tyrosine residue has been identified by sequence studies using radioactively labeled p-diazobenzoate and was found to be most probably Tyr-222 . Diethyl pyrocarbonate reacts with a tyrosine residue in the active center other than Tyr-222; the former could not be identified . Sequence studies further showed that Cys-211 is also partially modified by p-diazobenzoate . In addition, the sequence of residues 343-345 was found to be Ser-Trp-Trp instead of the tentative assignment Ser-Tyr-Trp made earlier . The results are briefly discussed on the basis of the existing three-dimensional model of the enzyme.

J Appl Bacteriol, 1986 Jul, 61(1), 25 - 38
Numerical taxonomy of proteolytic psychrotrophs from Queensland raw milks; O'Connor RE et al.; Eighty-seven proteolytic psychrotrophic micro-organisms were isolated from 11 bulk milk supplies of two Queensland factories from different climatic regions, before and after storage at 4 degrees C for 7 d . These isolates together with 15 reference strains formed the basis of a numerical taxonomic study involving 81 attributes . All but six isolates were pseudomonads . The strains clustered into nine groups, of which one group consisted of four yeasts . One group, containing 39 isolates, was designated as Pseudomonas fluorescens biovar 1; three groups, containing 27 isolates, as Ps . fluorescens biovar 5; and one group, containing 10 isolates, as Ps . putida biovar A . This study showed that the proteolytic psychrotrophic microflora of the 11 milks supplying the two factories was substantially different and that the proteolytic flora of 7 d refrigerated milk could not be estimated by examining the flora before storage.

J Dairy Res, 1986 May, 53(2), 301 - 12
Determination of the extracellular lipases of Pseudomonas fluorescens spp . in skim milk with the beta-naphthyl caprylate assay; McKellar RC et al.; A method based on the hydrolysis of beta-naphthyl caprylate (beta-NC) has been developed for quantitating extracellular lipase from Pseudomonas fluorescens . The assay was extremely sensitive to skim milk (SM); as little as 0.02 ml raw SM in a 2.0 ml reaction mixture resulted in an apparent loss of 50% of the lipase activity . Activity improved 3-fold when trypsin (50 micrograms/ml) was included in the reaction mixture . When super-simplex optimization was used to determine the optimum levels of beta-NC, Na taurocholate (NaTC), SM/lipase mixture and trypsin for maximum activity, NaTC was found to be unnecessary for activity . Subsequent addition of 15 mM-NaTC resulted in 80% loss of activity . On the other hand, NaTC was required for native lipase activity in the presence of SM . Native lipase was completely inhibited by heating at 70 degrees C for 2 min, while B52 lipase retained 75% of its activity under the same conditions . The assay was able to detect lipase produced by Ps . fluorescens B52 in SM at 5 degrees C when the cell density exceeded 10(8) colony forming units/ml . The presence of butterfat (3.5%) in the SM assay inhibited B52 lipase by 97% . The beta-NC assay gave results comparable to the tributyrin agar diffusion assay using cell-free extracts of ten strains of common dairy psychrotrophs . The results suggest that the beta-NC assay may be useful for determining lipase activity in raw SM.

Arch Biochem Biophys, 1986 May 1, 246(2), 622 - 32
Adenylylation state of glutamine synthetase and permeability properties of Pseudomonas fluorescens; Meyer JM et al.; The state of adenylylation, n, of glutamine synthetase (GS) in Pseudomonas fluorescens has been determined as a function of growth conditions . Compared to the behavior of Escherichia coli, atypical responses to either carbon or nitrogen starvation were observed when P . fluorescens was grown with either succinate, malate, or fumarate as the sole source of carbon and energy . Under conditions of carbon starvation (high NH4+, low dicarboxylic acid substrate), the value of n falls rapidly from 10 to 1.0 during prolonged incubation in the stationary phase, whereas the value of n is unexpectedly high (ca . 10) in extracts of nitrogen-starved cells . These abnormal responses are attributable to particular permeability properties of P . fluorescens cells compared to E . coli . The unusual changes in nitrogen-starved cells are related to the release of alpha-ketoglutarate by such cells during incubation or washing procedures . These changes can be prevented by the addition of cetyltrimethylammonium bromide (CTAB) to the cultures 5 min prior to harvesting the cells, or by freezing the cell pellets just after centrifugation and sonication within 3 min of suspension in buffer, or by suspending freshly harvested cells in buffer containing alpha-ketoglutarate and orthophosphate (i.e., effectors that favor deadenylylation of glutamine synthetase) . The abnormal changes which occur during carbon starvation in the presence of excess NH4+ can be prevented by addition of ATP and glutamine to the buffer in which the freshly harvested cells are suspended prior to sonication . The results suggest that during the stationary phase of growth on succinate, fumarate, or malate (but not on glucose), the cellular membrane becomes permeable to small molecules that regulate the adenylylation cascade, and indeed, it was observed that such whole cells expressed, without any chemical or physical treatment, more than 50% of the glutamine synthetase activity they contained . Such cells may be useful in studies to examine the effects of multiple metabolites on the regulation of glutamine synthetase adenylylation in situ.

Antimicrob Agents Chemother, 1986 Mar, 29(3), 488 - 95
Characterization of an antibiotic produced by a strain of Pseudomonas fluorescens inhibitory to Gaeumannomyces graminis var . tritici and Pythium spp; Gurusiddaiah S et al.; The production, isolation, and characterization of an antibiotic substance from cultures of Pseudomonas fluorescens 2-79 (NRRL B-15132) is described . P . fluorescens 2-79 originally was isolated from the roots of wheat and is suppressive to the wheat root disease take-all caused by Gaeumannomyces graminis var . tritici . The antibiotic was isolated from potato glucose broth cultures of strain 2-79 by solvent extraction . It was purified by silica gel column chromatography and was a greenish yellow, needle-shaped crystal with a melting point of 242 degrees C (decomposition) . It was soluble in methylene chloride, chloroform, acetone, 2 N sodium hydroxide, and 2 N hydrochloric acid and was insoluble in water, methanol, ethyl acetate, tetrahydrofuran, diethyl ether, carbon tetrachloride, hexane, and petroleum ether . On the basis of UV, infrared, 1H-nuclear magnetic resonance, 13C-nuclear magnetic resonance, mass spectral analysis, and elemental analysis, the structure of the antibiotic is proposed to be a dimer of phenazine carboxylic acid . Lithium aluminum hydride reduction of the antibiotic yielded hydroxymethyl phenazine as a major product which retained most of the biological characteristics of the parent molecule . There were no toxic symptoms when mice received this antibiotic by oral doses up to 464 mg/kg . The antibiotic showed excellent activity against several species of fungi, including the wheat pathogens Gaeumannomyces graminis var . tritici, Rhizoctonia solani, and Pythium aristosporum; and it may have a role in suppression of take-all in vivo by strain 2-79.

J Dairy Sci, 1986 Mar, 69(3), 658 - 64
Determination of the extracellular and cell-associated hydrolase profiles of Pseudomonas fluorescens sp . using the Analytab API ZYM system; McKellar RC; The extracellular and cell-associated hydrolase profiles of a number of Pseudomonas fluorescens strains were examined with the Analytab API ZYM system . Esterase/lipase was the only strong extracellular enzyme activity detected (mean 3.33): weak esterase, lipase, and leucine aminopeptidase activities were found with some strains (mean activities of 1.08, 1.53, and 1.40, respectively) . Very strong leucine aminopeptidase activity (4.5) was associated with the cells . Cell-associated trypsin, esterase/lipase, acid phosphatase, and phosphoamidase were also found . Neither extracellular nor cell-associated hydrolase profiles changed significantly when cells were grown in skim milk or mineral salts medium at either 5 or 20 degrees C . Similarly, added calcium did not seem required for synthesis of any of the enzymes . The extracellular enzyme profiles differed considerably from those of the cell-associated enzymes for all strains tested . An extracellular proteinase-deficient mutant of strain 32A (RM14) failed to produce significant quantities of extracellular esterase/lipase activity . Production of cell-associated enzymes was unaffected by the mutation . These results suggest that the Analytab API ZYM system may be useful in identifying psychrotrophs isolated from milk.

Appl Environ Microbiol, 1986 Mar, 51(3), 559 - 61
Quantitative determination of infinite inhibition concentrations of antimicrobial agents; Marwan AG et al.; We developed a method to determine the infinite inhibition concentrations (IICs) of antimicrobial agents . This method was based on finding the relative effectiveness of an inhibitor at various concentrations . Benzoic acid and parabens were tested on Saccharomyces bayanus, Hansenula sp., and Pseudomonas fluorescens . The relative effectiveness values of these compounds were established . A plot of the inhibitor concentration versus the reciprocal of relative effectiveness was linear . The chi-axis intercept was the concentration of the inhibitor which gave infinite microbial inhibition . For S . bayanus the IICs were 330, 930, 480, and 220 ppm (330, 930, 480, and 220 ml/liter) for benzoic acid and methyl-, ethyl-, and propylparabens, respectively . For Hansenula sp . the IIC was 180 ppm for benzoic acid . For P . fluorescens the IICs were 1,310, 960, and 670 ppm for methyl-, ethyl-, and propylparabens, respectively . Our results indicated that the IIC is affected by the growth medium . The advantages and applications of this method are discussed.

J Bacteriol, 1986 Mar, 165(3), 696 - 703
Molecular cloning of genetic determinants for inhibition of fungal growth by a fluorescent pseudomonad; Gutterson NI et al.; Pseudomonas fluorescens HV37a inhibits growth of the fungus Pythium ultimum in vitro . Optimal inhibition is observed on potato dextrose agar, a rich medium . Mutations eliminating fungal inhibition were obtained after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine . Mutants were classified by cosynthesis and three groups were distinguished, indicating that a minimum of three genes are required for fungal inhibition . Cosmids that contain wild-type alleles of the genes were identified in an HV37a genomic library by complementation of the respective mutants . This analysis indicated that three distinct genomic regions were required for fungal inhibition . The cosmids containing these loci were mapped by transposon insertion mutagenesis . Two of the cosmids were found to contain at least two genes each . Therefore, at least five genes in HV37a function as determinants of fungal inhibition.

J Dairy Res, 1986 Feb, 53(1), 97 - 115
Physicochemical properties of proteinases from selected psychrotrophic bacteria; Mitchell GE et al.; The physicochemical properties of eight extracellular proteinases secreted by psychrotrophic bacteria of dairy origin have been studied . Seven of these proteinases were able to withstand ultra heat treatment (UHT) with D values at 140 degrees C ranging from 2 to 300 s . The six Pseudomonas fluorescens proteinases were glycoproteins of mol . wt 47000-49500 . The two Serratia marcescens proteinases, of mol . wt of 51000, did not contain carbohydrate but in other respects were similar to the Pseudomonas proteinases . The proteinases were inhibited by various metal chelators and all contained Ca and Zn in similar proportions . Their amino acid compositions were similar, with alanine as the N-terminal group, cysteine completely absent and very low levels of methionine . Isoelectric points ranged from 5.10 to 8.25 . Their physical and chemical properties enabled them to be classified as alkaline metalloendopeptidases . A similarity index (S delta n) was used to predict sequence homology between ten proteinases of known amino acid composition . Comparisons of S delta n of these proteinases showed only minor sequence differences except for those of Ps . fluorescens MC60 . Heat resistance could not be related wholly to similarities in protein sequence, but could be related both to the strength of stabilizing Ca2+-protein interactions and to the randomness inherent within the folding of the peptide chain.

J Dairy Res, 1986 Feb, 53(1), 117 - 27
A rapid colorimetric assay for the extracellular lipase of Pseudomonas fluorescens B52 using beta-naphthyl caprylate; McKellar RC; Conditions necessary for the determination of crude extracellular lipase activity from Pseudomonas fluorescens B52 using beta-naphthyl caprylate (beta-NC), an 8-carbon ester, as the substrate were examined . Maximum enzyme synthesis occurred at 20 degrees C in pyruvate mineral salts medium containing 1 mM-CaCl2 . Bile salts were necessary for enzyme activity; 6 mM-Na taurocholate or Na deoxycholate gave maximum activity but the latter compound was inhibitory at higher concentrations . Activity was optimal in N-Tris{hydroxymethyl}methyl-2-aminoethane sulphonic acid buffer pH 8.0 at 40 degrees C . A comparison of beta-NC with beta-naphthyl butyrate (a 4-carbon ester) and beta-naphthyl myristate (a 14-carbon ester) showed that beta-NC was the best substrate; Km values of 0.0415, 0.141 and 0.200 mM and Vmax values of 67.2, 20.1 and 5.28 mumol ml-1 h-1 were obtained for those substrates respectively . The enzyme was inhibited 50% in its activity against beta-NC by 0.009 mM-EDTA, 0.0007% (w/v) mixed alkyltrimethylammonium bromide and 0.00275% (w/v) Triton X-100 . The biochemical properties determined using beta-NC as substrate are consistent with those reported for the lipases of other strains of Ps . fluorescens using natural substrates.

Appl Environ Microbiol, 1986 Feb, 51(2), 418 - 21
Effects of cysteine on growth, protease production, and catalase activity of Pseudomonas fluorescens; Himelbloom BH et al.; Cysteine inhibits growth of and protease production by Pseudomonas fluorescens NC3 . Catalase activity in P . fluorescens NC3 was increased by cysteine . The addition of exogenous hydrogen peroxide did not increase catalase activity, thus suggesting a role for the endogenous generation of hydrogen peroxide via the autoxidation of cysteine.

Exp Parasitol, 1986 Feb, 61(1), 65 - 75
Trypanosoma cruzi: ultrastructural changes produced by an anti-trypanosomal factor from Pseudomonas fluorescens; Mercado TI et al.; Trypomastigotes and amastigotes of Trypanosoma cruzi exhibited distinct ultrastructural alterations when treated with an extracellular lytic substance (anti-trypanosomal factor) produced by Pseudomonas fluorescens . Marked swelling of the parasites and detachment of the plasma membrane from the subjacent cytoplasm were observed after 15 min of treatment . After 3 hr, the nucleus was extensively damaged, the kinetoplast was indistinguishable, the mitochondrion was markedly swollen, the cytoplasm was disrupted, and the plasma membrane showed extensive blebbing and focal loss of subpellicular microtubules . These changes were progressive, as shown by the occurrence of parasite ghosts after 10 hr . Amastigotes exhibited an extremely swollen mitochondrion with disrupted internal structure, widening of the perinuclear space, and blebbing of the external nuclear membrane . The kinetoplast, however, remained clearly discernible . The drugs used today in controlling Chagas' disease are toxic . Therefore, there is a need for new anti-trypanosomal agents such as the Pseudomonas fluorescens antibiotics . The observations described in this study indicate the potential chemotherapeutic usefulness of these compounds for this disease.

EMBO J, 1986 Feb, 5(2), 231 - 6
Ice nucleation activity of Pseudomonas fluorescens: mutagenesis, complementation analysis and identification of a gene product; Corotto LV et al.; A DNA fragment of 7.5 kb from Pseudomonas fluorescens MS1650 confers an ice nucleation phenotype when cloned in Escherichia coli . This DNA encodes a protein with an apparent mol . wt of 180 kd, which is found in both inner and outer membrane fractions of transformed E . coli cells . Insertion mutations throughout a 3.9-kb region cause deficiency in ice nucleation, and eliminate the 180-kd protein . Complementation is not observed between any pair of mutations, suggesting that the nucleating phenotype is encoded by a single transcriptional unit . Mutations in most parts of the 3.9-kb region are not completely deficient in phenotype: they still generate ice nuclei at low frequency . One insertion mutation was found to generate pseudowild revertants, which had undergone deletions of the entire insertion and some of the adjacent sequence; these could account for the incomplete deficiency . These deletions displayed depressed nucleation temperatures, but their nucleation frequencies were close to that of the wild-type gene.

Rev Argent Microbiol, 1986, 18(1), 45 - 8
{In vitro bacteriostatic activity of piperacillin sodium against Pseudomonas}; de Nobile VB et al.; One hundred and twenty-two strains of Pseudomonas isolated from diverse pathological processes were typified . In vitro activity of Piperacillin (antibiogram and MIC) was studied and compared with another two semisynthetic penicillins, Carbenicillin and Mezlocillin, and two aminoglycosides, Amikacin and Gentamicin . The strains isolated corresponded to: Pseudomonas aeruginosa (116), Pseudomonas cepacia (3), Pseudomonas fluorescens (1) and Pseudomonas putida (1) . It was found that 110 Pseudomonas aeruginosa, one Pseudomonas cepacia and one Pseudomonas fluorescens were susceptible to Piperacillin . The susceptibility to this drug was quite higher than to the other penicillins tested . The MIC50 and MIC90 of Piperacillin were lower than those of semisynthetic penicillins and the minimum susceptibility value . In comparison with aminoglycosides it was found that, although Amikacin and Gentamicin did not reach the minimum value, Piperacillin exhibited higher MICs.

J Appl Bacteriol, 1986 Jan, 60(1), 37 - 44
Possible role of calcium in the formation of active extracellular proteinase by Pseudomonas fluorescens; McKellar RC et al.; The requirement for calcium during synthesis of extracellular proteinase by Pseudomonas fluorescens B52 was examined . Synthesis was monitored using cells resuspended at high density in fresh growth medium . Optimum enzyme production was found with cells grown to mid-logarithmic phase in mineral salts medium containing calcium chloride (1.0 mmol/l) . Inhibition of synthesis by EDTA addition was rapid, similar to the effect produced by chloramphenicol, an inhibitor of translation . Appearance of enzyme initiated by calcium addition to depleted cells was rapid and was dependent on de novo protein synthesis . Sephadex G-100 chromatography of L-{4,5-3H}leucine-labelled cell-free supernatant liquids revealed that, in the absence of calcium, a low molecular weight (12000-14000 daltons) irreversibly inactive 'precursor' of the proteinase was formed . The results are consistent with the hypothesis that calcium is required for structural integrity of the proteinase as well as for activity.

Biochem J, 1985 Nov 1, 231(3), 651 - 4
Identification of the covalently bound flavins of D-gluconate dehydrogenases from Pseudomonas aeruginosa and Pseudomonas fluorescens and of 2-keto-D-gluconate dehydrogenase from Gluconobacter melanogenus; McIntire W et al.; An improved method is presented for the purification of 8 alpha-(N1-histidyl)riboflavin, 8 alpha-(N3-histidyl)riboflavin and their 2',5'-anhydro forms, which permits the isolation of sizeable quantities of each of these compounds from a synthetic mixture in pure form . Flavin peptides were isolated from the D-gluconate dehydrogenases of Pseudomonas aeruginosa and Pseudomonas fluorescens and from the 2-keto-D-gluconate dehydrogenase of Gluconobacter melanogenus . After conversion into the aminoacyl-riboflavin, the flavin in all three enzymes was identified as 8 alpha-(N3-histidyl)riboflavin . By sequential treatment with nucleotide pyrophosphatase and alkaline phosphatase, the flavin in each enzyme was shown to be in the dinucleotide form.

Mikrobiologiia, 1985 Sep-Oct, 54(5), 755 - 62
{Regulation of the synthesis of the key enzymes for naphthalene catabolism in Pseudomonas putida and Pseudomonas fluorescens carrying the biodegradation plasmids NAH, pBS3, pBS2 and NPL-1}; Starovoitov II; Regulation of the synthesis of key enzymes catalysing naphthalene catabolism was studied in Pseudomonas strains containing different plasmids of naphthalene biodegradation . The synthesis of naphthalene oxygenase, salicylate hydroxylase, catechol-1,2-oxygenase and cathechol-2,3-oxygenase was shown to be regulated in both the coordinated and non-coordinated manner.

Pediatr Infect Dis, 1985 Sep-Oct, 4(5), 508 - 12
Pseudobacteremia due to Pseudomonas fluorescens; Simor AE et al.; Pseudomonas fluorescens was recovered from 62 of 22,270 (0.26%) blood cultures, from 57 patients, over a 22-month period at a pediatric hospital . No illness was attributable to the blood culture isolate . A case-control study identified a significant correlation between the recovery of P . fluorescens in blood culture and concomitant coagulation studies (p less than 0.0001) . In all cases blood for coagulation studies had been obtained at the same time as the blood culture . A review of venipuncture technique revealed that occasionally the coagulation study tubes (containing 3.8% sodium citrate) were being inoculated before blood culture bottles . P . fluorescens was subsequently isolated from coagulation tubes and from sodium citrate solutions prepared and dispensed in the hospital for use in coagulation studies . In vitro studies confirmed that sodium citrate solutions supported the growth of P . fluorescens, with preferential growth at 25 degrees C and 4 degrees C . This is the first description of P . fluorescens as a cause of pseudobacteremia . Pseudobacteremia was attributed to cross-contamination of blood cultures following inoculation of contaminated citrated collection tubes.

J Dairy Sci, 1985 Aug, 68(8), 1902 - 9
Identification of proteolytic pseudomonads isolated from raw milk; Kwan KK et al.; Proteolytic pseudomonads isolated from raw milk were classified by numerical taxonomy . Unweighted pair-group average-linkage cluster analysis was used to cluster 49 bacterial strains, of which 26 were Pseudomonas species, as described in the Bergey's Manual of Determinative Bacteriology, based on 52 characters . The milk isolates resided in two clusters; one containing Pseudomonas fluorescens and the other Pseudomonas fragi . The isolates identified with Pseudomonas fluorescens hydrolyzed milk proteins and milk fat and produced phospholipase . The Pseudomonas fragi-like isolates hydrolyzed milk proteins and milk fat but did not produce phospholipase and fluorescent pigment . The hydrolytic characteristics of milk isolates showed that the nature of the substrate and conditions under which the test was conducted were critical.

J Antibiot (Tokyo), 1985 Jun, 38(6), 767 - 71
Activity of safracins A and B, heterocyclic quinone antibiotics, on experimental tumors in mice; Okumoto T et al.; Safracins A and B, new antibiotics produced by Pseudomonas fluorescens A2-2, were tested for antitumor activity against mouse tumors . Structurally, these antibiotics belong to the saframycin family of antibiotics, and safracin B is 21-hydroxysafracin A . They showed antitumor activity against L1210 and P388 leukemias and B16 melanoma . The toxic and effective doses of safracin B were much lower than those of safracin A . Safracin B also prolonged the life span of tumor-bearing mice to a greater extent than safracin A . These results indicate that the alpha-carbinolamine structure plays an important role in the antitumor action of this type of antibiotic . Both safracins were, however, ineffective when their administration route differed from that used for inoculating tumor cells.

J Hyg (Lond), 1985 Jun, 94(3), 269 - 77
Efficacy of low-concentration iodophors for germicidal hand washing; Stiles ME et al.; The efficacy of iodophor germicides containing different concentrations of available iodine against transient (inoculated) bacteria and the natural hand microflora was compared with chlorhexidine gluconate (2 and 4%) liquid detergent (Hibitane), non-germicidal soap and a tap water rinse . The tap water rinse was ineffective compared with all other treatments . Only 4% chlorhexidine gluconate liquid detergent and iodophor containing 0.75% available iodine were significantly better than the non-germicidal soap for reduction of transient bacteria, Escherichia coli and Pseudomonas fluorescens, that had been inoculated onto hands . These agents also caused a significant reduction in the number of 'natural' microorganisms released from hands after a standard 15 s hand wash . The low-concentration iodophor products and the product containing 2% chlorhexidine gluconate failed to give results significantly better than the non-germicidal control soap . Baird-Parker medium and standard aerobic plate counts were highly correlated (r = 0.82), so that for studies of Gram-negative bacteria inoculated onto hands as a transient microflora, counts on Baird-Parker medium give a reasonable indication of the natural (residual) hand microflora.

J Biol Chem, 1985 Apr 25, 260(8), 5098 - 104
Malonyl coenzyme A synthetase . Purification and properties; Kim YS et al.; Malonyl coenzyme A synthetase (EC 6.2.1.14) was induced in Pseudomonas fluorescens grown on malonate as a sole carbon source . This enzyme was purified, for the first time, over 30-fold by the combination of ammonium sulfate precipitation, Sephadex G-150 gel filtration, DEAE-Sephacel ion exchange chromatography, and hydroxylapatite chromatography . The purified enzyme, which had a specific activity of about 0.512 mumol/min/mg, appeared to be electrophoretically homogeneous . The molecular size of the enzyme was determined to be 98,000 Da which is composed of two 49,000-Da subunits . The optimum pH for the enzyme was 7.5 . Malonyl coenzyme A synthetase requires ATP, CoA, and Mg2+ for the full enzyme activity . With succinate or acetate, the synthetic rate of CoA derivative was 40% of that observed with malonate . The malonyl coenzyme A synthetase showed typical Michaelis-Menten kinetics for the substrate, malonate, ATP, and coenzyme A, from which the Km values were calculated to be 3.8 X 10(-4) M, 2 X 10(-3) M, and 10(-4) M and Vmax values to be 0.117 mumol/min/mg, 0.111 mumol/min/mg, and 0.142 mumol/min/mg, respectively . The purified malonyl coenzyme A synthetase was immunogenic in the rabbit and Ouchterlony double diffusion analysis revealed a single precipitant line with the enzyme . The antiserum inhibited the enzyme activity and the extent of inhibition was dependent on the amount of the serum added.

Biochem Int, 1985 Apr, 10(4), 627 - 31
Ester synthesis at extraordinarily low temperature of -3 degrees C by modified lipase in benzene; Takahashi K et al.; The lipoprotein lipase from Pseudomonas fluorescens was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine . The modified lipase in which 55% of the amino groups in the enzyme molecule were coupled with polyethylene glycol was found to be soluble in benzene and catalyzed the reactions of ester synthesis, ester exchange, aminolysis and ester hydrolysis in benzene . The modified lipase had an extraordinary temperature-dependency: enzymic activity for methyl laurate synthesis from methyl alcohol and lauric acid increased with decreasing temperature and attained the maximum at the extremely low temperature of -3 degrees C . The optimum temperature for hydrolysis of methyl laurate was as low as -4 degrees C.

Can J Microbiol, 1985 Mar, 31(3), 232 - 7
Volatiles of Pseudomonas aeruginosa and related species by automated headspace concentration--gas chromatography; Zechman JM et al.; The volatile metabolites of three strains of Pseudomonas aeruginosa and one strain each of Pseudomonas cepacia, Pseudomonas maltophilia, Pseudomonas fluorescens, and Pseudomonas putida were analyzed using an automated headspace concentrator incorporating a gas chromatograph . The procedure does not require sample preparation and automates the entire analytical sequence to yield reproducible profiles of volatile constituents . Gas chromatographic profiles of the volatile metabolites of each species were obtained using a 20-min concentration period and two fused silica capillary columns of different polarities . The production of headspace metabolites from trypticase soy broth was studied in relationship to culture incubation time and initial cell concentration . The volatiles identified after 24 h incubation consisted of 1-butanol, isopentanol, toluene, 1-undecene, 2-butanone, 2-heptanone, 2-nonanone, and 2-undecanone . Sufficient amounts of specific metabolites were produced after 5 h incubation to provide information of possible diagnostic value . In particular, all P . aeruginosa strains produced a distinctive series of 1-undecene and methyl ketones after 5 h incubation of media inoculated to provide 2 X 10(6) cells/mL . The results indicate that when growth and analytical conditions are held constant, P . aeruginosa and related species produce characteristic profiles of headspace metabolites . Since conventional bacteriological tests require 24 h or more for the identification of these pseudomonads, automated volatile analysis could provide an alternative means for the rapid detection of these bacteria.

J Dairy Res, 1985 Feb, 52(1), 91 - 100
Inhibition by chelating agents of the formation of active extracellular proteinase by Pseudomonas fluorescens 32A; McKellar RC et al.; The effect of chelating agents on extracellular proteinase production by Pseudomonas fluorescens 32A was examined . Increasing concentrations of orthophosphate slightly stimulated growth while inhibiting proteinase synthesis . Fifty per cent inhibition was found at 35 and 28 mM-orthophosphate at 5 and 20 degrees C respectively . Extracellular protein concentration was reduced by 30% when cells were grown with 100 mM-orthophosphate . Polyacrylamide gel electrophoresis of the cell-free supernatants suggested that reduced enzyme synthesis had taken place as evidenced by the decrease in staining intensity of the protein band corresponding to the proteinase . Other phosphate compounds could replace orthophosphate as an inhibitor . Extent of inhibition was related to chain length; polyphosphates with 4-6 or 13-18 phosphorus atoms were the most effective inhibitors . EDTA (0.5 mM) completely inhibited proteinase synthesis . This inhibition could be partly reversed by Ca2+ and, to a lesser extent, Mn2+ . Proteinase production at 5 degrees C in skim milk was completely inhibited by phosphate glass (P13-P18) . Control experiments showed that loss of activity with chelators was not due to inhibition of preformed enzyme . The results suggest a possible role for polyphosphates in controlling proteinase production in stored milk.

Appl Environ Microbiol, 1985 Feb, 49(2), 374 - 6
Phthalate metabolism in Pseudomonas fluorescens PHK: purification and properties of 4,5-dihydroxyphthalate decarboxylase; Pujar BG et al.; Pseudomonas fluorescens PHK uses 4,5-dihydroxyphthalate as the sole carbon source for o-phthalate catabolism . This intermediate is the substrate for a decarboxylase of the pathway yielding protocatechuate . The decarboxylase was purified to homogeneity by an affinity chromatography procedure in which the reaction product, protocatechuate, was used as a ligand . We describe some properties of the enzyme, including its apparent molecular weight of 420,000 as determined by gel filtration and of 66,000 after sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, consistent with a hexameric functional protein . The apparent Km for the substrate 4,5-dihydroxyphthalate was 10.4 microM . The characteristics of this enzyme are compared with those described for the isofunctional enzyme from P . testosteroni.

Mikrobiologiia, 1985 Jan-Feb, 54(1), 146 - 51
{Kinetic principles of the process of formaldehyde biodegradation}; Leonova VE et al.; The kinetics of formaldehyde biodegradation by Pseudomonas fluorescens was studied under the conditions of batch cultivation . The process was shown to depend on the substrate, biomass and metabolites concentrations . Their combined action is satisfactorily described by the equation (formula; see text) where Vmax = 3.23 g/g/h, Ks = 2.52 g/l and Kx = 10.2 g/l . The equation is adequate as was demonstrated in experiments in which the organism was cultivated under the continuous conditions on the formaldehyde-containing sewage from the production of antibiotics.

Zentralbl Mikrobiol, 1985, 140(2), 91 - 5
{Effect of inorganic detergent components on the biodegradation of alkyl benzenesulfonates}; Dimkov R et al.; The rate of biodegradation of the alkyl chain of alkylbenzene sulphonates (ABS) carried out of the alkyl chain by pure adapted cultures of Pseudomonas fluorescens and Aspergillus sp . has been examined in the presence of four anorganic test compounds of commercial detergents . The strains were isolated from sewages by elected cultures . It has been established that the sodium perborate gave a completely different effect on the microbial metabolism of ABS than that obtained with polyphosphate, sulphate and silicate . The perborate possesses an inhibitory effect on the growth and the surfactant-degradative ability of the strains . The authors concluded from it that the presence of 20% perborate in the formula of commercial detergents must be kept in mind where environmental problems are concerned.

Eur J Biochem, 1984 Dec 3, 145(2), 245 - 56
Chemical modification of sulfhydryl groups in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens . Involvement in catalysis and assignment in the sequence; van Berkel WJ et al.; The cysteine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens were modified with several cysteine reagents . One of the five sulfhydryl groups reacts rapidly and specifically with N-ethylmaleimide without inactivation of the enzyme . Cysteine-116 was found to be the reactive cysteine by isolation of a labeled tryptic peptide . The enzyme is easily inactivated by mercurial compounds . The original activity can be fully restored by treatment of the modified enzyme with sulfhydryl-containing compounds . The rate of incorporation of mercurial compounds is pH-independent and is pseudo-first-order up to 90-95% loss of activity . The reaction shows saturation kinetics . The substrate p-hydroxybenzoate protects the enzyme from fast inactivation . The mercurial compounds themselves inhibit the inactivation reaction at concentrations higher than 80 microM . A spin-labeled derivative of p-chloromercuribenzoate reacts fairly specifically with only Cys-152 on use of enzyme prelabeled with N-ethylmaleimide, in contrast to p-chloromercuribenzoate which reacts with additional cysteine residues, i.e . Cys-211 and Cys-158 . From these results it is concluded that modification of Cys-152 decreases drastically the affinity of the enzyme for the substrate . The results strongly indicate that the substrate binding site and Cys-152 are interdependent . This observation is not obvious when the three-dimensional data only are considered . The modified enzyme exhibits a somewhat higher affinity for NADPH than the native enzyme . Modification of N-ethylmaleimide-prelabeled enzyme by p-chloromercuribenzoate leads to absorbance difference spectra showing maxima at 250 nm, 290 nm and 360 nm . The intensities of the absorbance difference maxima at 290 nm and 360 nm are strongly dependent on the pH value of the solution . The intensities are very low at low pH values and increase with increasing pH values, reaching a maximum at about pH = 9 . The ionizing group shows a pK value of about 7.6 . The maximal molar difference absorption coefficient at 290 nm is 3200 M-1cm-1 at pH 9, suggesting that tyrosine residues ionize under the conditions of modification of the enzyme . The results are discussed in the light of the known three-dimensional structure.

Plasmid, 1984 Nov, 12(3), 181 - 8
Molecular characterization of a plasmid from Pseudomonas fluorescens involved in styrene degradation; Bestetti G et al.; In this paper evidence is given that in a strain of Pseudomonas fluorescens able to grow on styrene as the sole carbon source, the degradation pathway of styrene is inducible and plasmid dependent . The plasmid, which we have called pEG is self-transmissible between Pseudomonas strains and has a size of 37 kb . A restriction map has been constructed and evidence for an inducible transcription of two separate regions of the plasmid has been obtained.

Hoppe Seylers Z Physiol Chem, 1984 Nov, 365(11), 1345 - 50
A new metalloproteinase from Pseudomonas fluorescens biotype I; Diermayr P et al.; Isolation and purification of a metalloproteinase from Pseudomonas fluorescens Biotype I are described . The molecular mass of the enzyme is 46 kDa, its isoelectric point is 8.1, its activity is trypsin-like . The amino-acid composition of the single chain protein is given . One molecule of the enzyme contains 1 atom of zinc and 9 atoms of calcium.

Z Lebensm Unters Forsch, 1984 Oct, 179(4), 288 - 95
Heat inactivation of exogenous proteinases from Pseudomonas fluorescens . I . Possibility of inactivation in milk; Kroll S et al.; The inactivation reaction of the proteinase of a P . fluorescens strain of biotype I in milk was investigated at 130-150 degrees C, also in milk and in buffer with and without added CaCl2 at temperatures below 100 degrees C . The decline in activity corresponded to first order kinetics in the UHT region; Ea = 115 kJ/mol . D values were 290 (130 degrees C), 124 (140 degrees C) and 54 s (150 degrees C); therefore, the usual temperature time combinations of UHT treatment are not sufficient to achieve the required rates of inactivation . At temperatures below 80 degrees C, inactivation corresponded increasingly to second order kinetics with considerably higher reaction rates; at 55 degrees C, an inactivation reaction corresponding to that induced by UHT treatment could be achieved at a thermal stress lower by a factor of 500 . This "low temperature inactivation" was observed in a further 20 strains representing the spectrum of P . fluorescens . The average rates of inactivation following heat treatment in milk for 20 min are 47% at 55 degrees C and 44% at 60 degrees C . This can be regarded as the most effective temperature range for the inactivation of the proteinases in milk . Clear connections can be seen between the biotype groups and the optimum temperature for inactivation: biotype group I ca . 55 degrees C, group II (with a few exceptions) less than or equal to 50 degrees C and group III greater than or equal to 60 degrees C . The inactivation reaction is systematically influenced by the proteins and Ca++ ions present in milk.

J Biochem (Tokyo), 1984 Sep, 96(3), 815 - 20
Effect of Brij 58 on the hydrolysis of methyl butyrate by lipase from Pseudomonas fluorescens; Nakagawa A et al.; Purified Pseudomonas fluorescens lipase {EC 3.1.1.3} exhibited slight activity on water-soluble esters such as methyl butyrate, and this activity was increased on addition of Brij 58 (20 oxyethylene hexadecyl ether) to the solution . This stimulating effect of Brij 58 on hydrolysis of various esters (dimethyl succinate, butyl n-acetate, and tributyrin) in aqueous solution was unspecific . Hydrolysis of methyl butyrate depended on the molecular ratio of Brij 58 to lipase, being maximal (about 8 times the basal level at 37 degrees C with 80 mM substrate in 0.1 M NaCl solution) with 30 mol of Brij 58 per mol of lipase . Comparative studies showed that all polyoxyethylene (POE) alkyl ethers tested, stimulated the methyl butyrate hydrolyzing activity and that the Adekatol SO series (dihydric normal alcohol ethoxylate) also stimulated the appreciably active, whereas Triton X-100, sodium cholate, sodium deoxycholate, sodium dodecylsulfate, POE, and fatty acids had no effect . Comparison of the effects of Brij 58 on the methyl butyrate hydrolyzing activities of various lipolytic enzymes indicated that its effect was specific for this lipase . Brij 58 had no detectable effect with emulsified esters, such as supersaturated methyl butyrate and triolein.

Eur J Biochem, 1984 Aug 15, 143(1), 189 - 97
Rapid relaxation processes in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens revealed by subnanosecond-resolved laser-induced fluorescence; Visser AJ et al.; Time-resolved fluorescence studies were carried out on the FAD bound to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens . The transient fluorescence exhibits complex decay kinetics with at least a short lifetime component in the 50-500-ps time region and a longer one in the range 1.5-3.5 ns . The shorter-lifetime component has a larger contribution in the presence of substrate (p-hydroxybenzoate) or inhibitor (p-aminobenzoate) . The quenching of the fluorescence is both static and dynamic in nature . The decay of fluorescence anisotropy shows that the FAD environment is both flexible and rigid . The FAD mobility can be enhanced by dilution of the enzyme, by raising the temperature, or by the binding of substrate or inhibitors . The anisotropy results are interpreted in part in terms of a monomer-dimer equilibrium, whereby the FAD in the monomer contains much more flexibility . The above-mentioned effects induce a shift of the equilibrium to the monomeric side . From a constrained parameter fitting the dissociation constant is estimated to be about 1 microM for the free enzyme and somewhat higher for the binary complexes between the enzyme and substrate or inhibitor . pH variation has only a slight effect on fluorescence or anisotropy decay parameters, while dimethylsulfoxide appears to promote dissociation into monomers by weakening hydrophobic interaction between the subunits . The results are discussed in the light of newly developed insights into the functional role of rapid structural fluctuations in enzyme catalysis.

J Biochem (Tokyo), 1984 Aug, 96(2), 545 - 52
Purification, crystallization, and molecular properties of aspartase from Pseudomonas fluorescens; Takagi JS et al.; Aspartase {L-aspartate ammonia-lyase, EC 4.3.1.1} of Pseudomonas fluorescens was highly purified to homogeneity and crystallized . The purified enzyme sedimented as a monodisperse entity upon ultracentrifugation with a s0(20),w value of 8.6S . Upon polyacrylamide gel electrophoresis (PAGE), the enzyme migrated as a single band . The molecular weight of the native enzyme was 173,000 +/- 3,000, as determined by sedimentation equilibrium analysis, and that of the enzyme subunit was determined to be 50,000 +/- 1,500 by sodium dodecyl sulfate (SDS)-PAGE . Cross-linking experiments using dimethyl suberimidate followed by SDS-PAGE indicated that the native enzyme was composed of four subunits with identical molecular weight . The amino acid composition of the enzyme was determined.

J Neurochem, 1984 Aug, 43(2), 499 - 506
Malonate, malonyl-coenzyme A, and acetyl-coenzyme A in developing rat brain; Mitzen EJ et al.; Free malonate, malonyl-coenzyme A (malonyl-CoA), and acetyl-CoA were assayed in rat brain at developmental ages from the 20th day of gestation to 60 days of postnatal life . The determination of malonate was based on its conversion to malonyl-CoA and decarboxylation to acetyl-CoA by enzyme extracts from Pseudomonas fluorescens . The resulting acetyl-CoA reacted with {4-14C}oxaloacetate to form {5-14C}citrate, which was isolated by TLC . Malonyl-CoA in perchloric acid extracts from brain was converted to acetyl-CoA by rat liver mitochondrial malonyl-CoA decarboxylase (EC 4.1.1.9) . Acetyl-CoA derived from this step was assayed by a modified CoA-cycling procedure . Brain acetyl-CoA was also assayed by CoA cycling . Prenatal brain contained no free malonate but malonyl-CoA was present . The acetyl-CoA level was relatively high just prior to birth and declined slightly with growth . Malonate concentrations after birth rose rapidly to reach 192 nmol/g wet weight at 60 days . Adult levels for malonyl-CoA and acetyl-CoA were 1.83 and 1.90 nmol/g wet weight, respectively . The origin and natural role of free malonate in brain are not known but deacylation of malonyl-CoA by reversal of the malonyl-CoA synthetase reaction is postulated . Rat liver and kidney also contain substantial concentrations of free malonate.

J Biochem (Tokyo), 1984 May, 95(5), 1513 - 6
Spirulina ferredoxin-NADP+ reductase . The complete amino acid sequence; Yao Y et al.; The amino acid sequence of ferredoxin-NADP+ oxidoreductase {EC 1.18.1.2, FNR} from Spirulina sp., a blue-green alga, was determined . Spirulina ferredoxin-NADP+ oxidoreductase was composed of 294 amino acid residues and the molecular weight of the holoenzyme was 34,135 . An apparent homology of the amino(N)-terminal region was found between ferredoxin-NADP+ reductases from Spirulina and spinach . We also found some sequence similarities in human erythrocyte glutathione reductase and p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, both of which are NADPH-dependent FAD enzymes.

Biull Eksp Biol Med, 1984 May, 97(5), 565 - 7
{Comparison of the immunodepressive action of microbial deamidases from different sources}; Kozlov EA et al.; The role of glutaminase activity of microbial deamidases in the immunodepressant action of these enzymes was studied . Escherichia coli asparaginase, asparagin and glutamin deamidases from Pseudomonas fluorescens and Mycobacterium album were found to have an inhibitory effect on the PHA-stimulated lymphocyte blast transformation . The inhibitory activity of deamidases with the asparaginase-glutaminase ratios 1 : 1.5 and 1 : 1.3 was one order of magnitude higher than that of Escherichia coli asparaginase with the ratio 1 : 0.02 . It is assumed that glutaminase activity plays an essential role in the immunodepressant action of deamidases .

J Biochem (Tokyo), 1984 Apr, 95(4), 1047 - 54
Purification and some properties of intracellular esterase from Pseudomonas fluorescens; Nakagawa A et al.; A novel esterase was found in Pseudomonas fluorescens cells and purified to homogeneity as determined by polyacrylamide gel electrophoresis . The esterase was extracted from the cells by freeze-thawing and hypotonic treatment . Purification was achieved by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose and benzylamine-agarose and then electrophoresis . The enzyme catalyzed the hydrolysis of methyl esters, such as methyl butyrate, but its hydrolyzing activity decreased with increase in the chain length of the alcohol moiety, and it did not catalyze the hydrolysis of triacylglycerols, such as triacetin . In contrast, the enzyme acted on various acyl residues in a series of methyl esters, such as dimethyl succinate, methyl methacrylate, and dimethyl malate . The optimum pH for activity of this enzyme with methyl butyrate was 7.0-8.5 . The enzyme was inhibited by phenylmethylsulfonylfluoride . Its molecular weight was estimated as 48,000 by molecular sieve electrophoresis and gel filtration on Sephadex G-150.

Eur J Biochem, 1984 Mar 15, 139(3), 637 - 44
The importance of monopole-monopole and monopole-dipole interactions on the binding of NADPH and NADPH analogues to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens . Effects of pH and ionic strength; Wijnands RA et al.; NADPH binding to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens is found to be strongly dependent on pH and ionic strength . In the ionic strength range of 0.02-0.15 M, optimal NADPH binding is observed at a pH value of 6.4 . Extrapolation of the dissociation constants to infinite ionic strength shows that under these conditions optimal binding occurs at pH values greater than 8 . Similar results were obtained for complexes between the enzyme and two NADPH analogues in the presence or absence of the substrate . The experimental data can be explained by a theoretical model in which monopole-monopole or monopole-dipole interactions between the enzyme and the ligand are dominant . Changes in the former interaction prevail at low ionic strength and low pH values while the changes in the latter prevail at high ionic strength and high pH values . The dipole moment of the enzyme in the direction of the NADPH binding site was calculated from the ionic strength and pH dependence of the complex formation . The calculated dipole moment of the enzyme is about 2000 Debye at pH 6 and decreases to about 1100 Debye at pH 8.5 . The results are discussed with respect to published results, including data obtained from the enzyme from a different source.

Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1728 - 32
Nucleotide sequence of the tms genes of the pTiA6NC octopine Ti plasmid: two gene products involved in plant tumorigenesis; Klee H et al.; The nucleotide sequence of the tumor morphology locus, tms, from pTiA6NC has been determined . The sequence analysis indicates that each of two polyadenylylated transcripts encoded by this locus contains an open reading frame; the predicted transcript 1 gene product has a molecular size of 83,769 daltons, and the predicted transcript 2 gene product, of 49,588 daltons . The precise start and stop positions of the transcript 2 RNA have been mapped with S1 nuclease . Several insertion mutations have been constructed . One of these localizes the transcript 2 promoter within the 72 base pairs 5' to transcription initiation . Significant homology was observed between the protein encoded by transcript 1 and the adenine binding region of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, suggesting that the transcript 1 protein binds adenine either as substrate or cofactor.

Mikrobiologiia, 1984 Mar-Apr, 53(2), 271 - 4
{Growth kinetics of microorganisms with various ecological strategies in a dialysis culture at low specific growth rates}; Dorofeev AG et al.; A dialysis culture was found to be most suitable for studying the metabolism of microorganisms at a low specific growth rate . The biomass of all microorganisms studied in the dialysis culture increased with time in a linear fashion; hence, the energy spent for growth decreased in proportion to a decrease in the specific growth rate . Microorganisms growing in oligotrophous environment (Arthrobacter globiformis and Lipomyces tetrasporus ) spent much less energy comparing to microorganisms from eutrophic habitats (Pseudomonas fluorescens and Debaryomyces formicarius ).

Can J Microbiol, 1984 Mar, 30(3), 396 - 405
Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens; Lee WS et al.; Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures . Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P . fluorescens . Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments . Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions . The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar . Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures . The opposite effect was observed in carbon-limited cultures . When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected . While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures . This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures . In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected . Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme . Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source . Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures . The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.

Z Lebensm Unters Forsch, 1984, 178(3), 179 - 86
{Proteolytic activities of Ps . fluorescens in milk: determination with azocasein in comparison with HPA}; Kroll S et al.; Proteolytic activity of psychotrophie bacteria in milk can be measured in a practical and comparatively sensitive manner using Azocasein as a substrate . The necessary parameters were exemplarily developed and demonstrated with a typical, strongly proteolytic strain of Pseudomonas fluorescens (No . 112) . Comparison of the improved Azocasein method with a modified version of the HPA method of Cliffe and Law--they had proposed it for predicting the stability of UHT milk--showed nearly the same sensitivity with six test strains of Pseudomonas fluorescens . The first significant detection of proteolytic activity could be made approximately at the same time except with one strain of the biotype II (miscellaneous strains) . It is discussed, whether the demonstrated methods can be judged (by the bacterial counts of detection) as sufficiently sensitive for to prediction of term spoilage during storage of UHT products.

Am J Med, 1984 Jan, 76(1), 62 - 8
Pseudomonas fluorescens bacteremia from blood transfusion; Khabbaz RF et al.; In October 1980, two units of blood contaminated with Pseudomonas fluorescens caused septic transfusion reactions in two recipients at a Chicago hospital; one patient died . Both units had been purchased from the same blood center . Investigation at the blood center and at other hospitals it supplied revealed another fatal case of P . fluorescens sepsis that had occurred one year earlier . Despite extensive environmental culturing at the blood center, the source of P . fluorescens was not identified . However, comparison of the interval between collection and administration of contaminated and non-contaminated units indicated that prolonged storage was a risk factor that may have caused clustering of cases in one hospital . Laboratory studies showed that small inocula of P . fluorescens proliferated in refrigerated fresh whole blood and reached 10(6) to 10(7) colony-forming units per milliliter seven days after incubation . These data suggest that prolonged storage of blood may be an important risk factor for the development of transfusion-related sepsis.

J Antibiot (Tokyo), 1983 Oct, 36(10), 1279 - 83
Safracins, new antitumor antibiotics . I . Producing organism, fermentation and isolation; Ikeda Y et al.; Safracins, new antibiotics with a novel skeleton, were discovered in a culture broth of Pseudomonas sp . The producing organism has been identified as Pseudomonas fluorescens . Safracins A and B were isolated by ethyl acetate extraction and chromatography on silica gel.

Biull Eksp Biol Med, 1983 Sep, 96(9), 83 - 4
{Effect of glutamin(asparagin)ase preparations from microorganisms on DNA synthesis in tumor cells}; Pekhov AA et al.; The effect of glutamin (asparagin)ase from Pseudomonas fluorescens and Pseudomonas boreopolis 526 on DNA synthesis by tumor cells, lines CaOv and L8, has been studied . The L8 cells have been demonstrated highly sensitive to the enzyme.

J Bacteriol, 1983 Sep, 155(3), 1105 - 9
Chromate resistance plasmid in Pseudomonas fluorescens; Bopp LH et al.; Chromate resistance of Pseudomonas fluorescens LB300, isolated from chromium-contaminated sediment in the upper Hudson River, was found to be plasmid specified . Loss of the plasmid (pLHB1) by spontaneous segregation or mitomycin C curing resulted in a simultaneous loss of chromate resistance . Subsequent transformation of such strains with purified pLHB1 plasmid DNA resulted in a simultaneous re-acquisition of the chromate resistance phenotype and the plasmid . When pLHB1 was transferred by conjugation to Escherichia coli, the plasmid still conferred chromate resistance.

Appl Environ Microbiol, 1983 Aug, 46(2), 333 - 7
Heat-stable protease from Pseudomonas fluorescens T16: purification by affinity column chromatography and characterization; Patel TR et al.; A heat-stable extracellular protease from Pseudomonas fluorescens T16, a psychrotroph, was purified by affinity column chromatography on a carbobenzoxy-D-phenylalanine-triethylene tetramine-Sepharose-4B column . The purified enzyme is a monomer with a molecular weight of 38,905 +/- 2,000 . In an analytical ultracentrifuge, the Schlieren profile revealed a single symmetrical peak . The sedimentation coefficient was estimated to be 3.93S . Alpha-casein was the preferred substrate, with a Km of 0.05 mM . Heating crude enzyme and purified enzyme in buffer at 50, 90, and 120 degrees C resulted in a rapid initial loss of more than 50% of the initial activity followed by a gradual inactivation which exhibited first-order kinetics . The activation energy for the hydrolysis of casein was calculated to be 3.2 kcal/mol (13.4 kJ/mol).

J Dairy Res, 1983 Aug, 50(3), 365 - 74
Growth of an extracellular proteinase-deficient strain of Pseudomonas fluorescens on milk and milk proteins; Torrie JP et al.; An extracellular proteinase-and lipase-deficient mutant of a psychrotroph, Pseudomonas fluorescens strain 32A, has been isolated and the absence of the proteinase enzyme confirmed by growth on differential media, enzyme assay and polyacrylamide gel electrophoresis . Competition between the parent and the mutant was observed when equal numbers of the 2 strains were inoculated together into raw skim-milk at 6 degrees C . Bitterness was detected at 6 degrees C in pasteurized skim-milk inoculated with the parent cells concurrent with the detection of proteolytic activity . In the case of the mutant, slight bitterness which did not increase with increasing cell numbers was detected in the absence of proteolysis . Mutant cells failed to grow on Na caseinate as the sole source of carbon . It was concluded that the extracellular proteinase, while not essential for growth in milk, does provide a selective advantage to the producer organism . This enzyme is, however, essential for growth on milk proteins and contributes to the development of bitterness in pasteurized milk.

Biochem Int, 1983 Jul, 7(1), 115 - 22
On the inhibition of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by adenosine nucleotides and metal ion complexes; Muller F et al.; The flavoprotein p-hydroxybenzoate hydroxylase is inhibited by adenosine nucleotides, Cibacron blue, phosphate ions and metal ion complexes . The inhibition of the enzyme is competitive with respect to NADPH . The most potent inhibitors of the enzyme are Cibacron blue and the metal ion complexes whereas the inhibition by the adenosine nucleotides is comparable to that by halogen ions . Some inhibitors cause quenching of the fluorescence emission of the protein-bound prosthetic group or perturb the absorption spectrum of the enzyme in the visible region allowing determination of the dissociation constant of the interaction between the free or the substrate-complexed enzyme and the inhibitors . The inhibition constants are in good agreement with the dissociation constants.

Appl Environ Microbiol, 1983 Jul, 46(1), 98 - 105
Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244; Hou CT et al.; NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria . Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography . The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases . Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis . The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein . It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol . Primary alcohols and diols were also oxidized, but at a lower rate . Alcohols with more than six carbon atoms were not oxidized . The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively . The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca . 34 kJ) . The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol . The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline . The enzyme oxidized the stereoisomers of 2-butanol at an equal rate . Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C) . It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.

Appl Environ Microbiol, 1983 Jul, 46(1), 6 - 12
Heat-stable proteases from psychrotrophic pseudomonads: comparison of immunological properties; Jackman DM et al.; A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column . The homogeneous enzyme preparation was used to prepare antiserum in rabbits . The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk . The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic determinants in proteases from different isolates . Rabbit antiserum to the purified protease gave precipitin bands with antigens (proteases) from 10 different isolates . However, the same antiserum did not inhibit the protease activity in cell extracts of isolates T10, T13, and T24 . By determining serological cross-reactions, proteases from psychrotrophic pseudomonads were shown to be different from one another.

Appl Environ Microbiol, 1983 Jun, 45(6), 1802 - 7
Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa; Askeland RA et al.; Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic . Maximum cyanogenesis by two strains of P . fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9 . Cyanide production per cell was optimum at 300 mM phosphate . A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM . The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase . Radioactive tracer experiments with {1-14C}glycine and {2-14C}glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P . fluorescens and P . aeruginosa . Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains . Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.

Eur J Biochem, 1983 Jun 1, 133(1), 91 - 108
p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens . 1 . Completion of the elucidation of the primary structure; Hofsteenge J et al.; As a final step in the elucidation of the primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, the amino acid sequences of a CNBr peptide (CB1, positions 111-276), that accounts for the middle part of the sequence, and the C-terminal CNBr peptide (CB2, positions 277-394) from the enzyme were determined . Important sequence information was obtained from two subfragments that were formed by the cleavage with CNBr of the Met-Thr sequence (positions 346-347) in peptide CB2 . The alignment of the two subfragments from peptide CB2 and three one-residue overlaps between peptides from one of these subfragments were confirmed by investigation of well-resolved parts of a 0.25-nm electron-density map . The sequence of residues 343-346 could not be determined with chemical methods and was assigned from the size and shape of the amino acids in the electron-density map . An important tool in the analysis of the amino acid sequence of peptide CB1 was the proteinase Lys-C from Lysobacter enzymogenes, which preferentially cleaves at lysine residues.

Eur J Biochem, 1983 Jun 1, 133(1), 109 - 18
p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens . 2 . Fitting of the amino-acid sequence to the tertiary structure; Weijer WJ et al.; The complete primary and tertiary structure of p-hydroxybenzoate hydroxylase is now known . The amino acid sequences of the two largest CNBr peptides have been fitted to the electron-density map at 0.25-nm resolution . The parts of the polypeptide chain contributing the residues to the FAD-binding site and the residues of the substrate-binding site have been identified . The active site is located in a large hydrophobic area enclosed by all domains of the enzyme structure . Here the substrate, p-hydroxybenzoate, is bound near, but not in direct contact with, the isoalloxazine ring system of FAD . Many side chains from the C-terminal part of the polypeptide chain are involved in subunit-subunit interactions . In the center of one of the largely hydrophobic contact areas between the subunits, a cluster of six aromatic amino acids was found.

Eur J Biochem, 1983 May 16, 132(3), 651 - 5
Purification and properties of aryl acylamidase from Pseudomonas fluorescens ATCC 39004; Hammond PM et al.; Aryl acylamidase has been purified from a strain of Pseudomonas fluorescens ATCC 39004, selected from soil on the basis of its ability to utilise acylanilide compounds as a sole source of carbon . The enzyme was purified to homogeneity by a combination of ion-exchange, hydrophobic and gel-permeation chromatography . A relative molecular mass of about 52 500 was estimated by gel filtration . The native enzyme was shown to be a monomeric protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis . The enzyme was maximally active at a pH of 8.6 and at a temperature of 45 degrees C . The enzyme shows Michaelis-Menten kinetics; Km values for nitroacetanilide (69 microM) and hydroxyacetanilide (6.1 microM) were low, indicating that the enzyme has a very high affinity for both substrates.

Mikrobiologiia, 1983 May-Jun, 52(3), 360 - 4
{Molecular DNA-DNA hybridization in Pseudomonas fluorescens and Pseudomonas putida}; Kiprianova EA et al.; Taxonomic analysis was carried out and DNA-DNA homology studied in cultures of subgroups formed by numerical classification of 124 Pseudomonas fluorescens and P . putida strains . The GC content in the DNA of strains belonging to P . fluorescens subgroups varied from 61.2 to 64.5%, and their DNA-DNA homology with the type strain P . fluorescens ATCC 13525 was 24 to 83% . The lowest genome relatedness with the type culture was found in biotype C of P . fluorescens and P . aureofaciens strains . The GC content in the DNA of strains belonging to two subgroups formed by numerical classification of P . putida varied from 63.8 to 65.0%, and the homology of their DNA with the DNA of the type strain of P . fluorescens was 0 to 10% (for strains of the first and second subgroups, respectively).

J Dairy Res, 1983 May, 50(2), 171 - 84
Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36; Stepaniak L et al.; A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0.1 M-phosphate buffer, pH 6.6, being completely denatured above 70 degrees C in 1 min . It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 degrees C (minimum at 55 degrees C), but stability was very high above 80 degrees C in this buffer . D-values were determined at 10 degrees C intervals in the range 70-150 degrees C in SMS from which a Z value of 31.9 degrees C and an Ea of 8.82 X 10(4) J mol-1 were calculated; the half-life at 150 degrees C was 9 s . Instability at 55 degrees C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups . The extent of inactivation experienced at 80 degrees C was inversely related to the rate of heating to 80 degrees C, i.e . length of time spent in the neighbourhood of 55 degrees C . Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 degrees C, presumably due to substrate binding . Attempts to stabilize the enzyme at 55 degrees C by addition of EDTA or by adjusting the reaction pH to 4.2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented . Unlike the proteinase from Ps . fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 degrees C from 80, 100 or 150 degrees C, but did regain autolytic activity on cooling to below 50 degrees C to an extent dependent on the duration of holding at the lower temperature . It is suggested that on heating to approximately 55 degrees C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 degrees C.

Eur J Biochem, 1983 Apr 5, 131(3), 527 - 33
Why are two different types of pyridine nucleotide transhydrogenase found in living organisms?
Voordouw G, van der Vies SM, Themmen AP.
Two types of pyridine nucleotide transhydrogenases have been reported in living organisms . The energy-linked transhydrogenase is found in mitochondria and in certain heterotrophic and photosynthesizing bacteria, while the non-energy-linked transhydrogenase is found in certain heterotrophic bacteria . The presence of a structurally similar non-energy-linked transhydrogenase in Azotobacter vinelandii, Pseudomonas aeruginosa and Pseudomonas fluorescens is readily shown in extracts from these bacteria with Western (protein) blotting . This non-energy-linked enzyme is lacking in Escherichia coli, while the presence of a structurally similar energy-linked enzyme in E . coli and in beef heart mitochondria is indicated with the Western blotting technique . Spinach (Spinacia oleracea) lacks the non-energy-linked transhydrogenase occurring in bacteria . The chloroplast enzyme ferredoxin:NADP+ oxidoreductase, which exhibits non-energy-linked transhydrogenase activity, is immunologically distinct from the bacterial transhydrogenases . In order to provide a rationale for the distribution of the two types of pyridine nucleotide transhydrogenases, the steady-state degrees of reduction of the NADP(H) and NAD(H) pools in A . vinelandii (R'NADP(H) and R'NAD(H)) have been measured for cells metabolizing sucrose at a variable oxygen flux (phi O2) . It is found that the degree of reduction of the NADP(H) pool is always higher than that of the NAD(H) pool (R'NADP(H) greater than R'NAD(H)) except when phi O2 goes to zero (R'NADP(H) approximately equal to R'NAD(H)) . Comparison of these results with literature values indicates that the inequality R'NADP(H) greater than R'NAD(H) is always found in a membrane-enclosed compartment, irrespective of the type of transhydrogenase present . This allows an understanding of the function of the two types of pyridine nucleotide transhydrogenases in vivo . The physiological role of non-energy-linked transhydrogenase is to catalyze the reaction NADPH + NAD+ leads to NADP+ + NADH, that of energy-linked transhydrogenase to catalyze the reaction NADH + NADP+ leads to NADPH + NAD+ . Since at equilibrium R'NADP(H) approximately equal to R'NAD(H) the inequality R'NADP(H) greater than R'NAD(H) under steady-state conditions explains the energy requirement in the latter reaction . The dependence of the non-energy-linked transhydrogenase activity of ferredoxin:NADP+ oxidoreductase on R'NADP(H) is compared with that of A, vinelandii transhydrogenase . The results indicate that this activity is unlikely to be of physiological importance in plant chloroplasts.

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