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