Biochem J, 2002 Oct 1, 367(Pt 1), 13 - 8 A catalytic consensus motif for D-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens; Klimacek M et al.; Lys-295, Asn-300 and His-303 of D-mannitol 2-dehydrogenase from Pseudomonas fluorescens were mutated individually into alanine (K295A, N300A and H303A respectively) . Purified mutants displayed catalytic efficiencies for NAD(+)-dependent oxidation of D-mannitol 300-fold (H303A), 1000-fold (N300A) and approx . 400000-fold (K295A) below the wild-type level . Comparison of primary kinetic isotope effects on kinetic parameters for D-fructose reduction by wild-type and mutants at pH 10.0 demonstrate that Asn-300 has an auxiliary role in stabilization of the transition state of hydride transfer, and His-303 contributes to substrate positioning . The large solvent isotope effect of 11+/-1 on k (cat) for mannitol oxidation by K295A at pH((2)H) 10.5 suggests a role for Lys-295 in general base enzymic catalysis . Positional conservation of Lys-295, Asn-300 and His-303 across a family of polyol-specific long-chain dehydrogenases suggests a unique catalytic signature: Lys-Xaa(4)-Asn-Xaa(2)-His (where 'Xaa' denotes 'any amino acid'). Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 483 - 7 Epub 2002 Jun 26. Cloning, functional expression and biochemical characterization of a stereoselective alcohol dehydrogenase from Pseudomonas fluorescens DSM50106; Hildebrandt P et al.; Sequencing of a genomic library prepared from Pseudomonas fluorescens DSM 50106 identified an orf showing 29% identity to a C alpha-dehydrogenase of Pseudomonas paucimobilis and high homology to several sequences with unknown functions derived from genome projects . The corresponding gene adhF1 encodes a dehydrogenase of 296 amino acids with a calculated molecular mass of 31.997 kDa . The gene was functionally expressed in E . coli using a rhamnose inducible expression system . The resulting recombinant enzyme was active in the pH range 6-10 (best pH 8) and at 5-25 degrees C . This dehydrogenase converts cyclic ketones to the corresponding alcohols utilizing the cofactor NADH . The highest activity was found for cyclohexanone . The enzyme also exhibits high stereoselectivity in the desymmetrization of the prochiral ketone acetophenone, producing optically pure ( R)-alpha-phenyl ethanol (>99%ee) at high conversion (95%). Water Environ Res, 2002 May-Jun, 74(3), 235 - 41 The role of kaolin particles in the performance of a carbamate-based biocide for water bacterial control; Pereira MO et al.; The influence of kaolin particles on the activity of Pseudomonas fluorescens and on the efficacy of a carbamate-based biocide was investigated . The results indicated that kaolin particles stimulated the activity of the bacteria for all buffered pH values studied (5, 7, and 9); this effect being more evident for the tests carried out at pH 5 and 9 . The presence of the clay in P . fluorescens suspensions decreased the efficiency of disinfection of the carbamate . The results also showed that kaolin was very effective in removing carbamate from the culture media . Therefore, the biocide concentration in the solution decreased, and consequently its availability to the bacteria was reduced . The results could be explained by the interaction between kaolin particles and carbamate and by the changes in the configuration of kaolin aggregates, depending on the pH value. Cryo Letters, 2000 Jan, 21(1), 5 - 12 Long-term retention of ice-nucleating active Pseudomonas fluorescens by overwintering colorado potato beetles; Castrillo LA et al.; Ice nucleating-active Pseudomonas fluorescens F264C was fed to Colorado potato beetles to determine bacterial retentioin in the beetle gut and its effect on the cold hardiness of this insect pest . The bacrterium was present in beetles recovered after overwintering in the field, seven months after their exposure to P . fluorescens . Retention was evident not only in the detection of the P . fluorescens ice nucleating gene, inaW, in bacterial cultures from beetle guts but also in the elevated supercooling points of some treated beetles. Appl Environ Microbiol, 2002 Aug, 68(8), 4122 - 6 Different responses of pyoverdine genes to autoinduction in Pseudomonas aeruginosa and the group Pseudomonas fluorescens-Pseudomonas putida; Ambrosi C et al.; We investigated the regulation of the psbA and pvdA pyoverdine biosynthesis genes, which encode the L-ornithine N(5)-oxygenase homologues in Pseudomonas strain B10 and Pseudomonas aeruginosa PAO1, respectively . We demonstrate that pyoverdine(B10), as the end product of its biosynthetic pathway, is a key participant of the control circuit regulating its own production in Pseudomonas strain B10 . In P . aeruginosa PAO1, however, pyoverdine(PAO1) has no apparent role in the positive regulation of the pvdA gene. J Appl Microbiol, 2002, 93(2), 205 - 13 Enzymes from isolates of Pseudomonas fluorescens involved in food spoilage; Rajmohan S et al.; AIMS: Psychrotrophic Gram-negative bacteria, such as Pseudomonas species, pose a significant spoilage problem in refrigerated meat and dairy products due to secretion of hydrolytic enzymes, especially lipases and proteases . This study characterized the enzymes produced by strains of Pseudomonas fluorescens isolated from pasteurized milk . METHODS AND RESULTS: Thirty-seven isolates of Ps . fluorescens from skimmed, semiskimmed and whole milk were all shown to be proteolytic and lipolytic on casein and tributyrin agar, respectively . The highest level of protease production by one isolate, SMD 31, from skimmed milk was in minimal salts medium containing 1 mmol x l(-1) calcium chloride at 20 degrees C . The proteases belonged to the class of metallo-proteases, as there was no residual activity with 10 mmol x l(-1) EDTA . They were heat stable and retained activity even after treatment at 121 degrees C for 20 min . One protease of 45-48 kDa was detected in unconcentrated supernatant fluid samples but, in three isolates from different milk sources, five proteases with molecular masses between 28 and 48 kDa were detected on a 12% zymogram casein gel following ultrafiltration . Attempts to purify the lipases proved unsuccessful . CONCLUSIONS: The characteristics of the major protease of 45-48 kDa correspond to those of proteases described for other Pseudomonas species isolated from a range of environments . However, the smaller proteases have not been described previously . SIGNIFICANCE AND IMPACT OF THE STUDY: In the absence of ultrafiltration the presence of the minor protease species may be missed and they may act as contaminants of the major protease in unpurified or semipurified samples. Biochemistry, 2002 Aug 6, 41(31), 10158 - 65 Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects; Klimacek M et al.; Mannitol dehydrogenases (MDH) are a family of Zn(2+)-independent long-chain alcohol dehydrogenases that catalyze the regiospecific NAD(+)-dependent oxidation of a secondary alcohol group in polyol substrates . pH and primary deuterium kinetic isotope effects on kinetic parameters for reaction of recombinant MDH from Pseudomonas fluorescens with D-mannitol have been measured in H(2)O and D(2)O at 25 degrees C and used to determine the relative timing of C-H and O-H bond cleavage steps during alcohol conversion . The enzymatic rates decreased at low pH; apparent pK values for log(k(cat)/K(mannitol)) and log k(cat) were 9.2 and 7.7 in H(2)O, respectively, and both were shifted by +0.4 pH units in D(2)O . Proton inventory plots for k(cat) and k(cat)/K(mannitol) were determined at pL 10.0 using protio or deuterio alcohol and were linear at the 95% confidence level . They revealed the independence of primary deuterium isotope effects on the atom fraction of deuterium in a mixed H(2)O-D(2)O solvent and yielded single-site transition-state fractionation factors of 0.43 +/- 0.05 and 0.47 +/- 0.01 for k(cat)/K(mannitol) and k(cat), respectively . (D)(k(cat)/K(mannitol)) was constant (1.80 +/- 0.20) in the pH range 6.0-9.5 and decreased at high pH to a limiting value of approximately 1 . Measurement of (D)(k(cat)/K(fructose)) at pH 10.0 and 10.5 using NADH deuterium-labeled in the 4-pro-S position gave a value of 0.83, the equilibrium isotope effect on carbonyl group reduction . A mechanism of D-mannitol oxidation by MDH is supported by the data in which the partly rate-limiting transition state of hydride transfer is stabilized by a single solvation catalytic proton bridge . The chemical reaction involves a pH-dependent internal equilibrium which takes place prior to C-H bond cleavage and in which proton transfer from the reactive OH to the enzyme catalytic base may occur . Loss of a proton from the enzyme at high pH irreversibly locks the ternary complex with either alcohol or alkoxide bound in a conformation committed of undergoing NAD(+) reduction at a rate about 2.3-fold slower than the corresponding reaction rate of the protonated complex . Transient kinetic studies for D-mannitol oxidation at pH(D) 10.0 showed that the solvent isotope effect on steady-state turnover originates from a net rate constant of NADH release that is approximately 85% rate-limiting for k(cat) and 2-fold smaller in D(2)O than in H(2)O. FEBS Lett, 2002 Jul 17, 523(1-3), 23 - 8 Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC; Baysse C et al.; Pyoverdines are the main siderophores of fluorescent pseudomonads . They comprise a quinoline chromophore, a peptide chain, and a dicarboxylic acid or a dicarboxylic acid amide side chain . Each Pseudomonas species produces a pyoverdine with a different peptide chain . A cytochrome c biogenesis DeltaccmC mutant of Pseudomonas fluorescens ATCC 17400 produces multiple pyoverdine forms, showing differences at the level of the chromophore or the side chain . When grown in the presence of L-cysteine, DeltaccmC produces only ferribactin, a non-fluorescent precursor of pyoverdine, while addition of oxidized glutathione improves pyoverdine production . We suggest that the conversion of ferribactin to pyoverdine does not take place in the DeltaccmC mutant because of lack of oxidizing power in the periplasm. Physiol Plant, 2002 Aug, 115(4), 550 - 562 A gene encoding stellacyanin is induced in Capsicum annuum by pathogens, methyl jasmonate, abscisic acid, wounding, drought and salt stress; Kong HY et al.; A stellacyanin cDNA clone (CASLP1) was isolated from a pepper cDNA library from hypersensitive response (HR) lesions of leaves infected with an avirulent strain of Xanthomonas campestris pv . vesicatoria . The deduced amino acid sequences of CASLP1 are homologous to those of stellacyanins from cucumber, maize, pea and Arabidopsis . The CASLP1 mRNA was not constitutively expressed in all organs of pepper, but strongly induced and accumulated in pepper tissues infected with X . campestris pv . vesicatoria, Colletotrichum coccodes, Phytophthora capsici or C . gloeosporioides . In situ hybridization results revealed that CASLP1 transcripts were strongly localized in the phloem areas of vascular bundles in infected tissues of pepper stems and fruits . CASLP1 mRNA accumulation was found in lower pepper leaves infected by either virulent or avirulent strains of X . campestris pv . vesicatoria and non-pathogenic Pseudomonas fluorescens, but not in uninoculated upper leaves . Induction of this CASLP1 gene occurred in pepper leaves applied with methyl jasmonate (MeJA), but not with ethylene, salicylic acid, dl-beta-amino-n-butyric acid and benzothiadiazole . Accumulation of CASLP1 transcripts was locally or systemically induced in pepper leaves upon mechanical wounding and was activated in a MeJA-dependent manner . The CASLP1 transcript was also strongly induced in leaf and stem tissues after exposure of pepper plants to abscisic acid, salt and drought. Mol Plant Microbe Interact, 2002 Jul, 15(7), 662 - 71 Characterization of NADH dehydrogenases of Pseudomonas fluorescens WCS365 and their role in competitive root colonization; Camacho Carvajal MM et al.; The excellent-root-colonizing Pseudomonas fluorescens WCS365 was selected previously as the parental strain for the isolation of mutants impaired in root colonization . Transposon mutagenesis of WCS365 and testing for root colonization resulted in the isolation of mutant strain PCL1201, which is approximately 100-fold impaired in competitive tomato root colonization . In this manuscript, we provide evidence that shows that the lack of NADH dehydrogenase I, an enzyme of the aerobic respiratory chain encoded by the nuo operon, is responsible for the impaired root-colonization ability of PCL1201 . The complete sequence of the nuo operon (ranging from nuoA to nuoN) of P . fluorescens WCS365 was identified, including the promoter region and a transcriptional terminator consensus sequence downstream of nuoN . It was shown biochemically that PCL1201 is lacking NADH dehydrogenase I activity . In addition, the presence and activity of a second NADH dehydrogenase, encoded by the ndh gene, was identified to our knowledge for the first time in the genus Pseudomonas . Since it was assumed that low-oxygen conditions were present in the rhizosphere, we analyzed the activity of the nuo and the ndh promoters at different oxygen tensions . The results showed that both promoters are up-regulated by low concentrations of oxygen and that their levels of expression vary during growth . By using lacZ as a marker, it was shown that both the nuo operon and the ndh gene are expressed in the tomato rhizosphere . In contrast to the nuo mutant PCL1201, an ndh mutant of WCS365 appeared not to be impaired in competitive root tip colonization. Microbiology, 2002 Jul, 148(Pt 7), 2223 - 31 Legionella pneumophila genes that encode lipase and phospholipase C activities; Aragon V et al.; Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic protozoans and human macrophages . The type II protein secretion system of the Gram-negative Legionella organism promotes intracellular infection . A lipase activity and a p-nitrophenylphosphorylcholine (pNPPC) hydrolytic activity are two of the factors that are diminished in L . pneumophila type II secretion mutants . The Legionella lipase activity was found to include free fatty acid release from di- and triacylglycerol substrates, in addition to the previously reported cleavage of monoacylglycerol . In a number of other bacterial systems, the release of p-nitrophenol from pNPPC is due to a phospholipase C . In an attempt to identify exoproteins that potentiate intracellular infection, three genes were identified and mutated in L . pneumophila strain 130b that were predicted to encode either a secreted lipase or a phospholipase C . The first two genes, which were designated lipA and lipB, encoded proteins containing the lipase consensus sequence {LIV}-X-{LIVFY}-{LIVMST}-G-{HYWV}-S-X-G-{GSTAC} . Mutations in lipA in particular reduced supernatant activity against mono- and triacylglycerols . However, loss of lipA and/or lipB did not impair the ability of L . pneumophila to infect Hartmannella amoebae or U937 cell macrophages . The third L . pneumophila gene, which was denoted plcA, encoded a protein that was highly homologous with a phospholipase C from Pseudomonas fluorescens . Inactivation of plcA diminished secreted pNPPC hydrolase activity but did not influence Legionella infection of host cells . Taken together, these data indicate that L . pneumophila has multiple lipases and possibly several phospholipase C enzymes but that LipA, LipB and PlcA are not among those exoproteins required for optimal intracellular infection. Appl Environ Microbiol, 2002 Jul, 68(7), 3597 - 605 Noninvasive quantitative measurement of bacterial growth in porous media under unsaturated-flow conditions; Yarwood RR et al.; Glucose-dependent growth of the luxCDABE reporter bacterium Pseudomonas fluorescens HK44 was monitored noninvasively in quartz sand under unsaturated-flow conditions within a 45- by 56- by 1-cm two-dimensional light transmission chamber . The spatial and temporal development of growth were mapped daily over 7 days by quantifying salicylate-induced bioluminescence . A nonlinear model relating the rate of increase in light emission after salicylate exposure to microbial density successfully predicted growth over 4 orders of magnitude (r(2) = 0.95) . Total model-predicted growth agreed with growth calculated from the mass balance of the system by using previously established growth parameters of HK44 (predicted, 1.2 x 10(12) cells; calculated, 1.7 x 10(12) cells) . Colonization expanded in all directions from the inoculation region, including upward migration against the liquid flow . Both the daily rate of expansion of the colonized zone and the population density of the first day's growth in each newly colonized region remained relatively constant throughout the experiment . Nonetheless, substantial growth continued to occur on subsequent days in the older regions of the colonized zone . The proportion of daily potential growth that remained within the chamber declined progressively between days 2 and 7 (from 97 to 13%) . A densely populated, anoxic region developed in the interior of the colonized zone even though the sand was unsaturated and fresh growth medium continued to flow through the colonized zone . These data illustrate the potential of a light transmission chamber, bioluminescent bacteria, and sensitive digital camera technology to noninvasively study real-time hydrology-microbiology interactions associated with unsaturated flow in porous media. Appl Environ Microbiol, 2002 Jul, 68(7), 3416 - 23 Antibiotic and biosurfactant properties of cyclic lipopeptides produced by fluorescent Pseudomonas spp . from the sugar beet rhizosphere; Nielsen TH et al.; Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp . To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp . from two different agricultural soils by using three different growth media . CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil . Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups . The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety . Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI . In vitro analysis using both purified CLPs and whole-cell P . fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi . The CLP-producing P . fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum. Appl Environ Microbiol, 2002 Jul, 68(7), 3226 - 37 Differential ability of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains to colonize the roots of pea plants; Landa BB et al.; Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp . that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases . Over 300 isolates of 2,4-DAPG-producing fluorescent Pseudomonas spp . were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to Fusarium wilt or take-all, respectively . Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers . Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants . Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)(-1) over the entire 15-week experiment . Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains. FEMS Microbiol Lett, 2002 Jun 4, 211(2), 195 - 201 Diversity of culturable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T . borchii mycelial growth; Sbrana C et al.; Isolation and physiological and molecular characterisation of culturable bacterial strains belonging to actinomycetes, pseudomonads and aerobic spore-forming bacteria were carried out on mycorrhizal root tips of Quercus robur var . peduncolata infected by Tuber borchii . Cellular density of the three bacterial groups in ectomycorrhizal root tips was estimated to be 1.3+/-0.11 x 10(6) cfu g(-1) dry weight for total heterotrophic bacteria and 1.08+/-0.6 x 10(5) (mean+/-S.E.), 1.3+/-0.3 x 10(5) and 1.4+/-0.2 x 10(5) cfu g(-1) dry weight for pseudomonads, actinomycetes and spore-forming bacteria respectively . Identification of pseudomonads by the Biolog system indicated, besides the most represented species Pseudomonas fluorescens (biotypes B, F and G), the occurrence of strains belonging to Pseudomonas corrugata . Amplified ribosomal DNA restriction analysis of actinomycetes and spore formers revealed at least three and six different groups of patterns, respectively . Many bacterial isolates were able to induce variations in growth rates of T . borchii mycelium; among these, 101 strains showed antifungal activity, whereas 17 isolates, belonging to spore formers, were able to increase mycelial growth up to 78% when compared to uninoculated mycelial growth . The potential role of these populations in the development and establishment of mycorrhizas is discussed. Plant J, 2002 Jan, 29(1), 11 - 21 Characterization of Arabidopsis enhanced disease susceptibility mutants that are affected in systemically induced resistance; Ton J et al.; In Arabidopsis, the rhizobacterial strain Pseudomonas fluorescens WCS417r triggers jasmonate (JA)- and ethylene (ET)-dependent induced systemic resistance (ISR) that is effective against different pathogens . Arabidopsis genotypes unable to express rhizobacteria-mediated ISR against the bacterial pathogen Pseudomonas syringae pv . tomato DC3000 (Pst DC3000) exhibit enhanced disease susceptibility towards this pathogen . To identify novel components controlling induced resistance, we tested 11 Arabidopsis mutants with enhanced disease susceptibility (eds) to pathogenic P . syringae bacteria for WCS417r-mediated ISR and pathogen-induced systemic acquired resistance (SAR) . Mutants eds4-1, eds8-1 and eds10-1 failed to develop WCS417r-mediated ISR, while mutants eds5-1 and eds12-1 failed to express pathogen-induced SAR . Whereas eds5-1 is known to be blocked in salicylic acid (SA) biosynthesis, analysis of eds12-1 revealed that its impaired SAR response is caused by reduced sensitivity to this molecule . Analysis of the ISR-impaired eds mutants revealed that they are non-responsive to induction of resistance by methyl jasmonate (MeJA) (eds4-1, eds8-1 and eds10-1), or the ET precursor 1-aminocyclopropane-1-carboxylate (ACC) (eds4-1 and eds10-1) . Moreover, eds4-1 and eds8-1 showed reduced expression of the plant defensin gene PDF1.2 after MeJA and ACC treatment, which was associated with reduced sensitivity to either ET (eds4-1) or MeJA (eds8-1) . Although blocked in WCS417r-, MeJA- and ACC-induced ISR, eds10-1 behaved normally for several other responses to MeJA or ACC . The results indicate that EDS12 is required for SAR and acts downstream of SA, whereas EDS4, EDS8 and EDS10 are required for ISR acting either in JA signalling (EDS8), ET signalling (EDS4), or downstream JA and ET signalling (EDS10) in the ISR pathway. Mol Plant Microbe Interact, 2002 Jun, 15(6), 567 - 76 Deleterious impact of a virulent bacteriophage on survival and biocontrol activity of Pseudomonas fluorescens strain CHAO in natural soil; Keel C et al.; Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases . One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect . We have isolated a lytic bacteriophage (phi)GP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains . phiGP100 was found to be a double-stranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb . Replication of phiGP100 was negatively affected at temperatures higher than 25 degrees C . phiGP100 had a negative impact on the population size and the biocontrol activity of P . fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms . In the presence of phiGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold . As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished . In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a phiDGP100-resistant variant of strain CHA0-Rif . To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment. Folia Microbiol (Praha), 2002, 47(2), 121 - 9 Induction of phenylpropanoid metabolism by Pseudomonas fluorescens against tomato spotted wilt virus in tomato; Kandan A et al.; Pseudomonas fluorescens (two native strains, one collection strain and their strain mixtures in all possible combinations) when applied through seed, seedling dip, soil and on leaf significantly reduced the tomato spotted wilt virus (TSWV) disease . In P . fluorescens-treated plants, the peroxidase and phenylalanine ammonia-lyase activity increased . Accumulation of phenolic compounds and lignin were shown to be increased in the P . fluorescens-treated plants . Isoperoxidase native PAGE indicated that the peroxidase isoforms in tomato plants induced by fluorescent pseudomonads were different from the control plants; this suggests that the general phenylpropanoid pathway is probably stimulated in tomato plants treated which in turn led to significant reduction in TSWV. Mem Inst Oswaldo Cruz, 2002 Apr, 97(3), 359 - 62 Oviposition attractancy of bacterial culture filtrates: response of Culex quinquefasciatus; Poonam S et al.; Oviposition attractants could be used for monitoring as well as controlling mosquitoes by attracting them to lay eggs at chosen sites . In the present study, culture filtrates of seven bacterial species were tested for their attractancy against gravid females of Culex quinquefasciatus . When their oviposition active indices (OAI) were studied, the culture filtrates of Bacillus cereus and Pseudomonas fluorescens exhibited oviposition attractancy (OAI = > 0.3) at 100 ppm and the OAI were respectively 0.70 and 0.47 . Culture filtrates of B . thuringiensis var . israelensis (wild type), B . t . var . israelensis (mutant) and B . sphaericus showed attractancy at 2000 ppm with OAI of respectively 0.71, 0.59 and 0.68 . However, the OAI of B . megaterium as well as Azospirillum brasilense was 0.13 (at 2000 ppm), which was less than 0.3 required to be considered them as attractants . When the oviposition attractancy of the bacterial culture filtrates were compared with that of a known oviposition attractant, p-cresol (at 10 ppm), the culture filtrates of B . t . var . israelensis (wild type) and B . cereus were found to be more active than p-cresol, respectively with 64.2 and 54.3% oviposition. Appl Environ Microbiol, 2002 Jun, 68(6), 2745 - 53 Siderophore typing, a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads; Meyer JM et al.; A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains . As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum, Pseudomonas fuscovaginae, Pseudomonas jessenii, Pseudomonas mandelii, Pseudomonas monteilii, "Pseudomonas mosselii," "Pseudomonas palleronii," Pseudomonas rhodesiae, "Pseudomonas salomonii," Pseudomonas syringae, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups . In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level . The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization . The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata, Pseudomonas frederiksbergensis, Pseudomonas graminis, and Pseudomonas plecoglossicida, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another . Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level. Ukr Biokhim Zh, 2001 Sep-Oct, 73(5), 90 - 4 {Inducible surface proteins from some strains of Pseudomonas species bacteria while adapting to cobalt}; Gruzina TG et al.; The possibility of increasing resistance of some Pseudomonas strains to cobalt at adaptation to monotonous increasing its concentration was studied . Strains Pseudomonas fluorescens B5242 and Pseudomonas fluorescens B894 are capable to increase its resistance in such conditions via inducible synthesis of protective surface proteins . The molecular masses of such proteins were 55.0; 45.0 and 33.0 kDa for P . fluorescens B5242 strain. J Agric Food Chem, 2002 Jun 5, 50(12), 3522 - 6 Antimicrobial and bactericidal activities of esters of 2-endo-hydroxy-1,8-cineole as new aroma chemicals; Miyazawa M et al.; Fourteen kinds of alkyl esters of 2-endo-hydroxy-1,8-cineole were synthesized, with yields of 57.8-98.0% . Each ester had a characteristic and unique odor . Especially, the tert-butyl acetate of 2-endo-hydroxy-1,8-cineole had the most interesting odor of all the synthetic esters . The antimicrobial and bactericidal activities of these synthetic esters against test bacteria (Staphylococcus aureus, Escherichia coli, and Pseudomonas fluorescens) were examined using the broth dilution method . As a result, the tert-butyl acetate of 2-endo-hydroxy-1,8-cineole showed the highest antimicrobial and bactericidal activities against all kinds of the test bacteria. Microb Ecol, 2001 Feb, 41(4), 360 - 368 Construction and Use of Broad Host Range Mercury and Arsenite Sensor Plasmids in the Soil Bacterium Pseudomonas fluorescens OS8; Petanen T et al.; We have generated new sensors for the specific detection and studies of bioavailability of metals by engineering Pseudomonas fluorescens with reporter gene systems . One broad host range mercury (pTPT11) and two arsenite (pTPT21 and pTPT31) sensor plasmids that express metal presence by luminescence phenotype were constructed and transferred into Escherichia coli DH5a and Pseudomonas fluorescens OS8 . The maximal induction was reached after 2 h of incubation in metal solutions at room temperature (22 degrees C) . In optimized conditions the half maximal velocity of reaction was achieved at acidic pH using a d-luciferin substrate concentration that was nearly sixfold lower for P . fluorescens OS8 than for E . coli DH5a . When using a luciferin concentration (150 mM) that was optimal for E . coli the luminescence declined rapidly in the case of Pseudomonas, for which the substrate level 25 mM gave a stable reading between about 20 min and 3 h . The ability of the strain OS8 to quantitatively detect specific heavy metals in spiked soil and soil extracts is as good, or even better in being a real-time reporter system, than that of a traditional chemical analysis . The Pseudomonas strain used is an isolate from pine rhizosphere in oil and heavy metal contaminated soil . It is also a good humus soil colonizer and is therefore a good candidate for measuring soil heavy metal bioavailability. Microb Ecol, 2001 Feb, 41(4), 290 - 300 Monitoring Population Size, Activity, and Distribution of gfp-luxAB-Tagged Pseudomonas fluorescens SBW25 during Colonization of Wheat; Unge A et al.; Increasingly, focus has been directed towards the use of microorganisms as biological control agents to combat fungal disease, as an alternative to chemical fungicides . Pseudomonas fluorescens SBW25 is one bacterial strain that has been demonstrated to promote plant growth by biocontrol of pathogenic fungi . To understand the mode of action of this bacterium, information regarding its localization and metabolic activity on plants is important . In this study, a gfp/luxAB-tagged derivative of P . fluorescens SBW25, expressing the green fluorescent protein (GFP) and bacterial luciferase, was monitored during colonization of wheat starting from seed inoculation . Since bacterial luciferase is dependent on cellular energy reserves for phenotypic expression, metabolically active cells were detected using this marker . In contrast, the stable GFP fluorescence phenotype was used to detect the cells independently of their metabolic status . The combination of these two markers enabled P . fluorescens SBW25 cells to be monitored on wheat plants to determine their specific location and metabolic activity . Studies on homogenized wheat plant parts demonstrated that the seed was the preferred location of P . fluorescens SBW25 during the 65-day time period studied, but the leaves and roots were also colonized . Interestingly, the bacteria were also found to be metabolically active on all plant parts examined . In situ localization of P . fluorescens SBW25 using a combination of different microscopic techniques confirmed the preference for the cells to colonize specific regions of the seed . We speculate that the colonization pattern of P . fluorescens SBW25 can be linked to the mechanism of protection of plants from fungal infection. Microb Ecol, 2001 Aug, 42(2), 193 - 200 Effect of 2,4-Diacetylphloroglucinol Producing, Overproducing, and Nonproducing Pseudomonas fluorescens F113 in the Rhizosphere of Pea; Naseby DC et al.; Pseudomonas fluorescens F113lacZY and modified strains carrying different function modifications were assessed for their impact in the rhizosphere of pea . Strain F113lacZY naturally produces the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phl) useful in plant disease control . The first modified strain of F113 was repressed in production of Phl, creating the Phl negative strain F113G22 . The second was a plasmid based overproducer of Phl (F113Rif (pCUGP)) . Both the F113lacZY and the F113Rif (pCUGP) strains increased the rhizoplane fungal populations, whereas the same strains reduced the rhizosphere soil fungal populations with respect to the control . Similar results were found with the rhizoplane and rhizosphere soil bacterial populations . The F113G22 treatment resulted in a significantly greater indigenous fluorescent Pseudomonas population than the F113lacZY and F113Rif (pCUGP) treatments and a greater total Pseudomonas population than the control, F113lacZY, and F113Rif (pCUGP) treatments . Overproduction of Phl did not affect the establishment of the introduced Pseudomonas population . None of the inocula displaced the indigenous populations, but the F113G22 inocula had an additive effect on the total Pseudomonas population . P (phosphatase), S (sulphatase), and N (urease) cycle enzyme activities were increased while C (glucosidase, NAGase) cycle activities were decreased by the F113lacZY and F113Rif (pCUGP) treatments, suggesting C leakage from the roots . Overall, most effects of inoculation compared to the wild type were found with the non-Phl-producing strain . Overproduction of Phl had little environmental effect in relation to wild-type inocula. Microb Ecol, 2001 Oct, 42(3), 458 - 465 Effect of Plant Species on the Kinetics of Conjugal Transfer in the Rhizosphere and Relation to Bacterial Metabolic Activity; Schwaner NE et al.; Conjugal transfer of a derivative of the RP4 plasmid between Pseudomonas fluorescens AS12 and Serratia plymuthica RF7 was compared in the rhizosphere of pea, wheat, and barley and related to the metabolic activity of the bacteria . To obtain a reliable measure of transfer, which allowed comparison of results between experiments, mathematical mass-action models were used to determine plasmid intrinsic kinetic coefficients . The data showed that not only were the rhizospheres highly conducive of transfer, with rates up to six orders of magnitude higher than in bulk soil, but differences between rhizospheres were also observed . Highest intrinsic kinetic coefficients were found in the pea rhizosphere (1.1-4.1 x 10-11), followed by the barley rhizosphere (2.4-7.2 x 10-12) and the wheat rhizosphere (2.2-2.9 x 10-13) . It was further shown that the metabolic activity of the cells in the rhizosphere of the three plants was not significantly different, and that activity and transfer were not correlated . Thus, the data demonstrated species specific rhizosphere effects on the conjugal transfer process that could not be attributed to different metabolic activities of the bacteria. Microb Ecol, 2001 Oct, 42(3), 438 - 445 Pseudomonas fluorescens DR54 Reduces Sclerotia Formation, Biomass Development, and Disease Incidence of Rhizoctonia solani Causing Damping-Off in Sugar Beet; Thrane C et al.; Effects of the biocontrol strain, Pseudomonas fluorescens DR54, on growth and disease development by Rhizoctonia solani causing damping-off in sugar beet were studied in soil microcosms and in pot experiments with natural, clay-type soil . In pot experiments with P . fluorescens DR54-treated seeds, significantly fewer Rhizoctonia-challenged seedlings showed damping-off symptoms than when not inoculated with the biocontrol agent . In the rhizosphere of P . fluorescens DR54 inoculated seeds, the bacterial inoculant was present in high numbers as shown by dilution plating and immunoblotting . By the ELISA antibody technique and direct microscopy of the fungal pathogen grown in soil microcosms, it was shown that the presence of P . fluorescens DR54 on the inoculated seeds had a strong inhibitory effect on development of both mycelium biomass and sclerotia formation by R . solani . In the field experiment, plant emergence was increased by treatment with P . fluorescens DR54 and the inoculant was found to be the dominating rhizosphere colonizing pseudomonad immediately after seedling emergence. Appl Biochem Biotechnol, 2002 Spring, 98-100, 963 - 76 Gas-phase enzymatic esterification on immobilized lipases in MCM-41 molecular sieves; Pires EL et al.; Several lipolytic enzymes were immobilized in the pores of MCM-41 and Al-MCM-41 molecular sieves and used as catalysts in the gas-phase esterification of acetic acid with ethanol . The entrapment of enzymes depended on the molecular sieve and the type of enzyme used . The order of enzymatic activity for enzymes entrapped in the pores of MCM-41 and Al-MCM-41 in the esterification reaction was OF (Rhizopus niveus lipases) < FAP-15 (Rhizopus oryzae lipases) < LEX (Mucorjavanicus lipases) < PS (Pseudomonas cepacia lipases) < AK (Pseudomonas fluorescens lipases) . Experiments carried out between 298 and 318 K showed no effect of temperature on catalyst yield, suggesting that the enzymes were appropriately immobilized in the pores of the molecular sieves, thus avoiding possible processes such as denaturing or autolysis. Appl Biochem Biotechnol, 2002 Spring, 98-100, 415 - 28 Purification and characterization of a microbial dehydrogenase: a vanillin:NAD(P)+ oxidoreductase; Bare G et al.; Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive . The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography . The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis . The native Mr value estimated by gel-filtration chromatography gave a value of 210,000 . The enzyme made use of NAD+ less effectively than NADP+ . Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD+ or NADP+ . The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound . The vanillin oxidation reaction was essentially irreversible . The pH optimum was 9.5 and the pI of the enzyme was 4.9 . Enzyme activity was not affected when assayed in the presence of salts, except FeCl2 . The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide . NAD+ and NADP+ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent . The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD+ or NADP+ . We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases. J Environ Biol, 2001 Jul, 22(3), 187 - 92 Role of bacteria in the epizootic ulcerative syndrome (EUS) of fishes; Mastan SA et al.; Bacteriological examination of certain water bodies and fishes carrying EUS was carried out . As a whole, 17 species of bacteria were isolated from the investigated water bodies and EUS affected fishes . The species of bacteria isolated from fishes are common to those isolated from water . Experimental infection trials conducted suggested that Aeromonas hydrophila in association with Pseudomonas fluorescens, may be playing the role of primary aetiological agent in producing EUS in fishes. Arch Latinoam Nutr, 2001 Dec, 51(4), 371 - 5 {Growth kinetics and proteases production of Pseudomonas fluorescens in raw milk at refrigeration}; Costa M et al.; The general use of refrigeration of raw milk has contributed to maintenance of its quality, but has induced the selection of a psychotrophic bacteria which during its growth produces heat-resistant enzymes responsible, in part, for the deterioration of long-life products . Given the condition of the prolonged refrigeration of the milk before the process, it was necessary to determine the growth curves for bacteria at temperatures between 2 degrees C and 10 degrees C and the kinetics of production of proteases in raw fresh milk, inoculated with Pseudomonas fluorescens RV1O, using an automatic fermentation system, with minimal agitation under conditions of controlled temperature and pH . The results show a development over 10(5) ufc/mL in the cultures at 6 degrees, 8 degrees and 10 degrees C during the first 30 h and proteasic activities over 30 microM p-NA/2 h getting levels of de 80-180 microM p-NA/2 h over 50 h of cultivation . Only the cultures at 2 degrees C appeared stable with inferior cell counts and without inducing enzymatic activity . At 4 degrees C an intermediate situation occurs. J Appl Microbiol, 2002, 92(6), 1078 - 86 Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists; Georgakopoulos DG et al.; AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps . corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum . METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp . was evaluated with dual cultures in various media . Pseudomonas strains inhibited the pathogen better than Bacillus strains . To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists . Four pairs of bacteria were selected . Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs) . Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments . In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists . Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo . Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off . Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier . CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet . Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation . SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides. J Bacteriol, 2002 Jun, 184(11), 3008 - 16 Characterization of interactions between the transcriptional repressor PhlF and its binding site at the phlA promoter in Pseudomonas fluorescens F113; Abbas A et al.; The phlACBD genes responsible for the biosynthesis of the antifungal metabolite 2,4-diacetylphloroglucinol (PHL) by the biocontrol strain Pseudomonas fluorescens F113 are regulated at the transcriptional level by the pathway-specific repressor PhlF . Strong evidence suggests that this regulation occurs mainly in the early logarithmic phase of growth . First, the expression of the phlF gene is relatively high between 3 and 13 h of growth and relatively low thereafter, with the phlACBD operon following an opposite expression profile . Second, the kinetics of PHL biosynthesis are specifically altered in the logarithmic phase in a P . fluorescens F113 phlF mutant . The phlA-phlF intergenic region presents a complex organization in that phlACBD is transcribed from a sigma(70) RNA polymerase-dependent promoter that is likely to overlap the promoter of the divergently transcribed phlF gene . The repression by PhlF is due to its interaction with an inverted repeated sequence, phO, located downstream of the phlA transcriptional start site . Cross-linking experiments indicate that PhlF can dimerize in solution, and thus PhlF may bind phO as a dimer or higher-order complex . Furthermore, it is now demonstrated that certain regulators of PHL synthesis act by modulating PhlF binding to phO . PHL, which has previously been shown to be an autoinducer of PHL biosynthesis, interacts with PhlF to destabilize the PhlF-phO complex . Conversely, the PhlF-phO complex is stabilized by the presence of salicylate, which has been shown to be an inhibitor of phlA expression. Waste Manag, 2002, 22(2), 195 - 200 Effect of microbial activity on the mobility of chromium in soils; Desjardin V et al.; The effect of microbial activity on the chemical state of chromium, in a contaminated soil located in the Rhjne-Alpes region (France), has been investigated . This soil contained 4,700 mg kg(-1) Cr, with about 40% present in the soluble hexavalent form . Indigenous microbial activity was found to significantly reduce Cr(VI) to the less mobile form (III) when the soil was incubated at 30 degrees C in an aqueous medium containing glucose and nutrients . A Cr(VI)-reducing strain of Streptomyces thermocarboxydus was isolated from the contaminated soil . The strain was found to metabolize Cr(VI) in a similar manner as an exogenous inoculum of Pseudomonas fluorescens LB300, and to precipitate chromium as a Cr oxyhydroxide with a gammaCrOOH-like local structure . The Cr(VI)-reducing activity of S . thermocarboxydus was induced, or significantly accelerated, by the aggregation of bacterial cells or their adhesion to suspended solid particles, and was stimulated in pure culture by glycerol and chromate. Microbiol Res, 2002, 157(2), 139 - 48 Horizontal and vertical movement of Pseudomonas fluorescens toward exudate of Macrophomina phaseolina in soil: influence of motility and soil properties; Singh T et al.; The role of motility and cell surface hydrophobicity in transport and dispersal of Pseudomonas fluorescens strains LAM1-hydrophilic, LAM2-hydrophobic and LAM(NM) (non-motile mutant of LAM2) under different soil conditions was studied . Maximum adhesion was recorded for LAM2 in clay loam (70%), followed by sandy loam (68%) and sandy soil (40%) . Vertical migration of P fluorescens isolates in soils was recorded at 5 and 25 cm flow of wafer or M . phaseolina exudate . In all the treatments, LAM1 exhibited maximum migration followed, by LAM2 and LAM(NM) . The rate of migration of such isolates was lowered in water irrigated soils compared to those irrigated with M . phaseolina exudate . In sandy soil, cells of LAM1 migrated up to 13 cm in comparison to LAM2 (11 cm) and LAN(NM) (9 cm) at 5 cm flow of fungal exudate . Population of LAM1, LAM2 and LAM(NM) was 5.7, 5.68 and 5.61 log cfu g(-1) soil at 1 cm depth, but it decreased to 2.56, 2.21 and 1.99 log cfu during migration up to 11 cm in sandy soil at 5 cm flow of fungal exudate . Greater motility was observed in sandy soil irrigated with water or fungal exudate, followed by sandy loam and clay loam . In general, filtration coefficient (lambda) of P . fluorescens was higher in soils irrigated with 5 cm of water or exudate than with 25 cm of irrigation . The horizontal movement of P . fluorescens strains in sandy soil adjusted at different psi m showed marked reduction with decrease in psi m . The non-motile LAN(NM) did not show chemotactic response and migrated up to a maximum of 3 mm in saturated soils (0 kPa) . After 96 h, LAM1 and LAM2 migrated upto 35 and 29 mm respectively in sandy soil . Motile isolates had significantly greater colonization of M . phaseolina sclerotia over the non-motile mutant. Curr Microbiol, 2002 Jun, 44(6), 396 - 400 Plant growth-promoting rhizobacteria-mediated induction of phenolics in pea ( Pisum sativum) after infection with Erysiphe pisi; Singh UP et al.; Qualitative and quantitative estimation of phenolic compounds was done through high performance liquid chromatography (HPLC) in different parts of pea ( Pisum sativum) after treatment with two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens (strain Pf4) and Pseudomonas aeruginosa (referred to here as Pag) and infection by Erysiphe pisi . The phenolic compounds detected were tannic, gallic, ferulic, and cinnamic acids on the basis of their retention time in HPLC . In all the treated plants, synthesis of phenolic compounds was enhanced . The induction of gallic, ferulic, and cinnamic acids was manyfold more than those in the control . Maximum accumulation of phenolic compounds was observed in plants raised from PGPR-treated seeds and infection with E . pisi . Under pathogenic stress, Pag performed better because a relatively higher amount of phenolics was induced compared with plants treated with Pf4. Biosci Biotechnol Biochem, 2002 Feb, 66(2), 430 - 3 Molecular cloning of the gene encoding thermostable endo-1,5-alpha-L-arabinase of Bacillus thermodenitrificans TS-3 and its expression in Bacillus subtilis; Takao M et al.; The gene that encodes a thermostable endo-arabinase (called ABN-TS) from Bacillus thermodenitrificans TS-3 was cloned, sequenced, and expressed in the mesophilic B . subtilis . The gene contained an open reading frame consists of 939 bp, which encodes 313 amino acids . The deduced amino acid sequence of the enzyme showed 50, 46, and 36% similarity with endo-arabinase from B . subtilis IFO 3134 (PPase-C), Pseudomonas fluorescens (ArbA), and Aspergillus niger (ABNA), respectively . The hydrophobic and acidic amino acids making up ABN-TS outnumbered those in PPase-C . The gene product expressed in B . subtilis, as the host, had substantially the same characteristics, and was stable up to 70 degrees C, and the reaction was optimal around 70 degrees C, as well as native ABN-TS. FEBS Lett, 2002 May 8, 518(1-3), 43 - 7 Identification of a Baeyer-Villiger monooxygenase sequence motif; Fraaije MW et al.; Baeyer-Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C-C bond using dioxygen and NAD(P)H . Using newly characterized BVMO sequences, we have uncovered a BVMO-identifying sequence motif: FXGXXXHXXXW(P/D) . Studies with site-directed mutants of 4-hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis . Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD-dependent monooxygenases: the so-called flavin-containing monooxygenases (FMOs), the N-hydroxylating monooxygenases (NMOs), and the BVMOs . Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F) . Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank. Acta Crystallogr C, 2002 May, 58(Pt 5), o268 - 71 Epub 2002 Apr 11. Pseudophomins A and B, a class of cyclic lipodepsipeptides isolated from a Pseudomonas species; Quail JW et al.; The crystal structures of pseudophomins A and B, with primary structures beta-hydroxydecanoyl-L-Leu-D-Glu-D-allo-Thr-D-Ile-D-Leu-D-Ser-L-Leu-D-Ser-L-Ile monohydrate, C(55)H(97)N(9)O(16).H(2)O, and beta-hydroxydodecanoyl-L-Leu-D-Glu-D-allo-Thr-D-Ile-D-Leu-D-Ser-L-Leu-D-Ser-L-Ile monohydrate, C(57)H(101)N(9)O(16).H(2)O, new cyclic lipodepsipeptides isolated from Pseudomonas fluorescens strain BRG100, have been solved . The absolute configuration of pseudophomin A has been determined from anomalous dispersion and the stereochemistry of the beta-hydroxy acid group is R. Appl Environ Microbiol, 2002 May, 68(5), 2229 - 35 Fusaric acid-producing strains of Fusarium oxysporum alter 2,4-diacetylphloroglucinol biosynthetic gene expression in Pseudomonas fluorescens CHA0 in vitro and in the rhizosphere of wheat; Notz R et al.; The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0 . FA production by 12 Fusarium oxysporum strains varied substantially . We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA'-'lacZ fusion . Only FA-producing F . oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression . The repressing effect of FA on phlA'-'lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF . One FA-producing strain (798) and one nonproducing strain (242) of F . oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil . F . oxysporum strain 798 (FA(+)) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA(-)) did not . In the phlF mutant CHA638, phlA expression was not altered by the presence of either F . oxysporum strain 242 or 798 . phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere. AIHA J (Fairfax, Va), 2002 Mar-Apr, 63(2), 178 - 83 UV disinfection of soluble oil metalworking fluids; Johnson DL et al.; The efficacy of a new high-intensity germicidal ultraviolet (UV) lamp for disinfection of opaque metalworking fluids (MWF) was investigated under laboratory conditions . Three dilutions of "soluble oil" MWF and water controls in a circulating system were inoculated with suspensions of Pseudomonas fluorescens to an initial concentration of about 10(7) colony forming units (CFU) per milliliter and irradiated with a submerged nonglass UV lamp . Aliquots of the circulating fluid were withdrawn before irradiation and at 10-sec intervals in the water control and 10-min intervals in the MWF . The samples were diluted with sterile water, plated, and counted after 18-24 hours' incubation . The UV-C radiation output of the lamp was estimated by irradiance measurements using a research radiometer . The concentration of CFU decreased by at least 2 logs (>99% reduction in culturability) in 30 sec in irradiated water . In all three dilutions of MWF, a 2-log decrease was obtained within 60 min . The UV-C output of the lamp was estimated at about 6 W . The disinfection appeared to follow a first order rate law both in MWF and in water . The CFU concentration was stable over time in unirradiated controls . These results demonstrate that UV disinfection is feasible in MWF opaque to both visible and UV wavelengths of light. Mol Biol Rep, 2001, 28(2), 63 - 72 Functional analysis of a chromosomal arsenic resistance operon in Pseudomonas fluorescens strain MSP3; Prithivirajsingh S et al.; We reported earlier about the detection of a chromosomally located arsenic operon (arsRBC) in a gram-negative bacterium Pseudomonas fluorescens strain MSP3, which showed resistance to elevated levels of sodium arsenate and sodium arsenite . The genes for arsenic resistance were cloned into the HindIII site of pBluescript vector producing three clones MSA1, MSA2 and MSI3 conferring resistance to sodium arsenate and arsenite salts . They were further sub-cloned to delineate the insert size and the sub-clones were designated as MSA11, MSA12 and MSI13 . The sub-clone pMSA12 (2.6 kb) fragment was further packaged into EcoRI-PstI site of M13mp19 and sequenced . Nucleotide sequencing revealed the presence of three open reading frames homologous to the arsR, arsB and arsC genes of arsenic resistance . Three cistrons of the ars operon encoded polypeptides ArsR, ArsB and ArsC with molecular weights ranging approximately 12, 37and 24 kDa, respectively . These polypeptides were visualized on SDS-PAGE stained with Coomassie blue and measured in a densitometer . The arsenic resistance operon (arsRBC) of strain MSP3 plasmid pMSA12 consists of 3 genes namely, arsR--encoding a repressor regulatory protein, arsB--the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells and arsC--a small cytoplasmic polypeptide required for arsenate resistance only, not for arsenite resistance . ArsB protein is believed to use the cell membrane potential to drive the efflux of intracellular arsenite ions . ArsC encodes for the enzyme arsenate reductase which reduces intracellular As(V) (arsenate) to more toxic As(III) (arsenite) and is subsequently extruded from the cell . The arsenate reductase activity was present in the soluble cytoplasmic fraction in E . coli clones . In the context of specified function of the arsenic operon encoded proteins, uptake and efflux mechanisms were studied in the wild strain and the arsenate/arsenite clones . The cell free filtrates of the arsenate clones (MSA11 and MSA12) obtained from P . fluorescens containing the arsC gene showed that arsenate reduction requires glutathione reductase, glutathione (GSH), glutaredoxin and ArsC protein . The protein was purified in an active form and a spectrophotometric assay was developed in which the oxidation of NADPH was coupled to reduction of arsenate . The molecular weights and the location of the polypeptides were obtained from Coomassie stained SDS-PAGE of extracellular and intracellular fractions of the cells. Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 363 - 76 Pseudomonas mosselii sp . nov., a novel species isolated from clinical specimens; Dabboussi F et al.; Twenty-two fluorescent pseudomonad strains of clinical origin received as Pseudomonas fluorescens (10 strains), Pseudomonasputida (10 strains) and Pseudomonas sp . (2 strains), and 33 type strains of the genus Pseudomonas were studied by numerical analysis based on 280 phenotypic characters . Twelve of the 22 clinical isolates clustered within a specific group, cluster IV . The other strains clustered within groups containing well-characterized fluorescent Pseudomonas species or did not cluster . Strains belonging to cluster IV were phenotypically different from all other clusters and subclusters of fluorescent pseudomonads . DNA-DNA hybridization showed that cluster IV corresponded to a genomic group sharing 72-100% DNA relatedness . DNA-DNA hybridization values with 67 strains representing 30 species of the genus Pseudomonas sensu stricto, including six recently described species (Pseudomonas veronii, Pseudomonas rhodesiae, Pseudomonas libanensis, 'Pseudomonas orientalis', 'Pseudomonas cedrella' and Pseudomonas monteilii), were below 49%, the value found for P . monteilii . The DNA G+C content of the type strain was 63 mol% . Comparison of the 16S rRNA gene sequence of a representative strain of cluster IV (CFML 90-83T) with sequences of other strains of the genus Pseudomonas revealed that strain CFML 90-83T was part of the P . fluorescens intrageneric cluster . On the basis of phenotypic, DNA-DNA hybridization and phylogenetic analyses, a novel species, Pseudomonas mosselii sp . nov., is proposed for the 12 strains of cluster IV . The type strain is P . mosselii CFML 90-83T (= ATCC BAA-99T = CIP 105259T) . The P . mosselii strains are phenotypically homogeneous and can be differentiated from other fluorescent species by several phenotypic features, including pyoverdine typing. Z Naturforsch {C}, 2002 Jan-Feb, 57(1-2), 9 - 16 The structures of the pyoverdins from two Pseudomonas fluorescens strains accepted mutually by their respective producers; Barelmann I et al.; From Pseudomonas fluorescens PL7 and PL8 structurally related pyoverdins were isolated and their primary structures were elucidated by spectroscopic methods and degradation reactions . Despite of some structural differences both Fe(III) complexes are taken up by either strain with a high rate . The implications regarding the recognition at the cell surface are discussed. Appl Environ Microbiol, 2002 Apr, 68(4), 2085 - 8 Inactivation of the regulatory gene algU or gacA can affect the ability of biocontrol Pseudomonas fluorescens CHA0 to persist as culturable cells in nonsterile soil; Mascher F et al.; Rifampin-resistant Pseudomonas fluorescens CHA0-Rif and mutants in which the regulatory gene algU (encoding sigma factor sigma(E)) or gacA (encoding a global regulator of secondary metabolism) was inactivated were compared for persistence in three nonsterile soils . Functional algU and (particularly) gacA were needed for CHA0-Rif to maintain cell culturability in soil. Appl Environ Microbiol, 2002 Apr, 68(4), 1962 - 71 Development and characterization of a green fluorescent protein-based bacterial biosensor for bioavailable toluene and related compounds; Stiner L et al.; A green fluorescent protein-based Pseudomonas fluorescens strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions . The biosensor is based on a plasmid carrying the toluene-benzene utilization (tbu) pathway transcriptional activator TbuT from Ralstonia pickettii PKO1 and a transcriptional fusion of its promoter PtbuA1 with a promoterless gfp gene on a broad-host-range promoter probe vector . TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (nptII) promoter . The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations . Our experiments using flow cytometry show that intermediate levels of gfp expression in response to toluene reflect uniform induction of cells . As the toluene concentration increases, the level of gfp expression per cell increases until saturation kinetics of the TbuT-PtbuA1 system are observed . Each inducer had a unique minimum concentration that was necessary for induction, with K(app) values that ranged from 3.3 +/- 1.8 microM for toluene to 35.6 +/- 16.6 microM for trichloroethylene (means +/- standard errors of the means), and maximal fluorescence response . The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di- and trichloroethylene, 2-methyl-2-butene) . The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline . Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample . These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants. Appl Environ Microbiol, 2002 Apr, 68(4), 1808 - 16 Method for spiking soil samples with organic compounds; Brinch UC et al.; We examined the harmful side effects on indigenous soil microorganisms of two organic solvents, acetone and dichloromethane, that are normally used for spiking of soil with polycyclic aromatic hydrocarbons for experimental purposes . The solvents were applied in two contamination protocols to either the whole soil sample or 25% of the soil volume, which was subsequently mixed with 75% untreated soil . For dichloromethane, we included a third protocol, which involved application to 80% of the soil volume with or without phenanthrene and introduction of Pseudomonas fluorescens VKI171 SJ132 genetically tagged with luxAB::Tn5 . For both solvents, application to the whole sample resulted in severe side effects on both indigenous protozoa and bacteria . Application of dichloromethane to the whole soil volume immediately reduced the number of protozoa to below the detection limit . In one of the soils, the protozoan population was able to recover to the initial level within 2 weeks, in terms of numbers of protozoa; protozoan diversity, however, remained low . In soil spiked with dichloromethane with or without phenanthrene, the introduced P . fluorescens VKI171 SJ132 was able to grow to a density 1,000-fold higher than in control soil, probably due mainly to release of predation from indigenous protozoa . In order to minimize solvent effects on indigenous soil microorganisms when spiking native soil samples with compounds having a low water solubility, we propose a common protocol in which the contaminant dissolved in acetone is added to 25% of the soil sample, followed by evaporation of the solvent and mixing with the remaining 75% of the soil sample. Microbiol Res, 2002, 157(1), 39 - 45 Effect of lacZY-marking of the 2,4-diacetyl-phloroglucinol producing Pseudomonas fluorescens-strain 5-2/4 on its physiological performance and root colonization ability; Alsanius BW et al.; Transgenic Pseudomonas fluorescens 5-2/4 with reinforced 2,4-diacetyl phloroglucinol (phl) production had shown increased biocontrol ability towards Pythium ultimum (Pu), but inferior root colonization ability compared to its wild type 5.014 . Therefore, enhanced root colonization ability of the transgenic strain by repeated inoculation and reisolation on tomato plants was suggested . As a preparation for repeated inoculation and reisolation cycles, the construction of a negative control of the transgenic strain 5-2/4 by marking with lacZY and screening for a mutant possessing qualities comparable to 5-2/4 was performed . Morphologically, colonies of all of the 11 selected mutants were similar on MLXgal medium . The root colonization ability of two of the lacZY-marked strains (mutants 1 and 10) was comparable to the parental strain . These were also able to compete with the resident microflora of tomato seedlings to the same extent as the parental strain . Five mutants were excluded due to lower growth rates on Yeast Malt, King's B Medium (KB) and 0.1 Tryptic Soy Agar (mutant 4, 5 and 8), excessive growth and higher siderophore production on KB (mutant 10) and increased protease production (mutant 2) . With respect to in vitro-antagonism of Pu, no differences could be found between the target strain and mutants 1, 3, 6, 7 and 9 . Examination of sole carbon source utilization of these five lacZY-marked strains revealed a significantly higher utilization of alpha-D-lactose and lactulose compared to 5-2/4 . However, significant differences could be found for 51% of the utilized carbon sources . Cluster analysis showed a high degree of similarity between 5-2/4 and mutant 1 both when analyzed with and without alpha-D-lactose . As mutant 1 also represented the colonization pattern most similar to the parental strain 5-2/4, it presents a presumptive subject for a negative control in the following inoculation and reisolation studies on tomato. Bioorg Khim, 2002 Jan-Feb, 28(1), 44 - 9 {Coprecipitation of the Pseudomonas fluorescens lipase with hydrophobic compounds as an approach to its immobilization for catalysis in nonaqueous media}; Gorokhova IV et al.; The precipitation of N-cetylamine, N-cetylacetamide, hexan-1,2-diol, cetyl alcohol, and poly(butyl metacrylate) in acetone-water media in the presence of the lipase from Pseudomonas fluorescens was found to be accompanied by the coprecipitation of the enzyme . Within the lyophilized coprecipitates, the lipase exhibits a high catalytic activity and enantioselectivity in the reaction of (1RS)-phenylethanol acetylation with vinyl acetate in t-butyl methyl ether . In order of increasing lipase activity, the coprecipitates can be arranged in the series: cetyl alcohol, poly(butyl metacrylate), hexadecane-1,2-diol, N-cetylamine, and N-cetylacetamide, with the activity 2.5- to 19-fold exceeding the activity of the native enzyme . The immobilization of the lipase on solid supports, such as Celite 545 (physical sorption) and Eupergit C250L (covalent binding), in the presence of hexadecane-1,2-diol was found to increase the esterifying activity of the enzyme . The English version of the paper. J Bacteriol, 2002 Mar, 184(6), 1587 - 96 Phenotypic selection and phase variation occur during alfalfa root colonization by Pseudomonas fluorescens F113; Sanchez-Contreras M et al.; During colonization of the alfalfa rhizosphere, Pseudomonas fluorescens F113 undergoes phenotypic variation, resulting in the appearance of colonies with different morphology . Among phenotypic variants, three isolates, C, F, and S were selected, with the C variant showing colony morphology identical to that of the inoculated wild-type strain and F and S having a translucent and diffuse morphology . Phenotypic variants F and S were shown to preferentially colonize distal parts of the roots and showed alterations in motility, swimming faster than the C variant and swarming under conditions that did not allow swarming of the C variant . The motility behavior correlated with overproduction of the fliC-encoded protein flagellin but not with hyperflagellation . Flagella of the F and S variants were several times longer than those of the C variant, and overproduction of flagellin was regulated at the transcriptional level . Variant F showed alterations in traits that have been shown to be important for rhizosphere colonization, such as siderophore, cyanide, and exoprotease production, and these phenotypes were complemented by a cloned gacA . Sequence analysis of the gacA alelle in variant F suggested selection of the phenotype in the rhizosphere . Variant F was also affected in other phenotypes, such as lipopolysaccharide structure and flocculation in unshaken liquid medium, which were not complemented by the gacA or gacS gene . Mutation of the F113 sss gene, encoding a site-specific recombinase, showed that most of the phenotypic variation was due to the activity of this recombinase, indicating that phase variation occurs during rhizosphere colonization. J Appl Microbiol, 2002, 92(3), 534 - 40 Isolation and identification of synthetic pyrethroid-degrading bacteria; Grant RJ et al.; AIMS: To isolate, select, identify and assess the potential for the biodegradation of synthetic pyrethroids (SPs) in sheep dips . METHODS AND RESULTS: SP-degrading bacteria were isolated from a mixed soil sample consisting of garden soil and soils from farms where SPs had been used . The two largest in size were then identified using microscopy, biochemical and genetic techniques to be members of the genera Pseudomonas and Serratia . By comparing the 16S rRNA gene sequences, the Pseudomonas sp . discovered was shown to group within the Pseudomonas fluorescens intrageneric cluster . The Serratia isolated was closely related to Serratia plymuthica . Cell growth and degradation was greatest in the Pseudomonas sp . culture where there was breakdown of 60 mg l(-1) to 6 mg l(-1) technical cypermethrin in 20 days . Tolerance to the SPs was greater in the Pseudomonas sp . but was found to depend on the availability of other carbon sources and nutrients . CONCLUSIONS: The bacteria characterized show the potential to be used in a bioremediation application for the treatment of SP residues . SIGNIFICANCE AND IMPACT OF THE STUDY: The SP-degrading bacteria may have use in the disposal of used SP residues and with further research could lead to an alternative route of disposal for use in agriculture or industry. J Paediatr Child Health, 2002 Feb, 38(1), 63 - 5 Pseudomonas fluorescens pseudobacteraemia: a cautionary lesson; Smith J et al.; OBJECTIVES: To describe an outbreak of pseudobacteraemia caused by Pseudomonas fluorescens in a paediatric population . To document and highlight the effect this outbreak had on clinical management and the steps taken to determine the source . METHODS: A clinical and microbiological investigation was carried out into a cluster of 38 pseudobacteraemias caused by Pseudomonas fluorescens in paediatric patients over a 10 month period . RESULTS: The source of the outbreak of pseudobacteraemia was found to be contaminated lithium heparin tubes, which were being filled prior to the filling of the blood culture bottle . Cultures of the same tubes yielded Pseudomonas fluorescens . As a result of the initial positive blood cultures, clinical management was altered in 18 cases . A staff education programme was instituted and eventually resulted in a cessation of the pseudobacteraemia . CONCLUSIONS: Pseudobacteraemias are a major cause of potentially inappropriate therapy in febrile children . Attention to detail in the collection of blood cultures can help reduce this outcome . Staff involved in the collection of blood cultures need to be aware of this potential source of contamination. Biosci Biotechnol Biochem, 2002 Jan, 66(1), 221 - 3 Preparation and catalytic performance of lipases encapsulated in sol-gel materials; Kato K et al.; Three kinds of lipases (from Candida antarctica, Pseudomonas cepacia, and Pseudomonas fluorescens) were encapsulated in inorganic matrices by the sol-gel method in order to synthesize chiral compounds by kinetic resolution . Sol-gel lipases prepared with vinyltriethoxysilane had higher hydrolysis activity for 2-octyl acetate than those with other silane precursors: tetramethoxysilane, methyltrimethoxysilane, and propyltrimethoxysilane. J Dairy Sci, 2002 Jan, 85(1), 19 - 27 Effects of high intensity pulsed electric field and thermal treatments on a lipase from Pseudomonas fluorescens; Bendicho S et al.; Milk and dairy products may contain microorganisms capable of secreting lipases that cause sensory defects and technological problems in the dairy industry . In this study, the effects of thermal and high-intensity pulsed electric field (HIPEF) treatments on an extracellular lipase from Pseudomonas fluorescens, suspended in a simulated skim milk ultrafiltrate (SMUF) have been evaluated . Heat treatments applied were up to 30 min from 50 to 90 degrees C . HIPEF treatments were carried out using pilot plant facilities in a batch or continuous flow mode, where treatment chambers consisted of parallel and coaxial configuration, respectively . Samples were subjected to up to 80 pulses at electric field intensities ranging from 16.4 to 37.3 kV/cm . This resulted in a lipase that was quite resistant to heat and also to HIPEF . High (75 degrees C-15 s) and low pasteurization treatments (63 degrees C-30 min) led to inactivations of 5 and 20%, respectively . Using the batch-mode HIPEF equipment, a 62.1% maximum activity depletion was achieved after 80 pulses at 27.4 kV/cm . However, when HIPEF treatments were applied in the continuous flow mode, an inactivation rate of just 13% was achieved, after applying 80 pulses at 37.3 kV/cm and 3.5 Hz . The results of both heat and HIPEF treatments on enzyme inactivation were adjusted with good agreement to a first-order kinetic model (R2 > 62.3%). Bioresour Technol, 2002 Mar, 82(1), 33 - 41 Solid state fermentation of broiler litter for production of biocontrol agents; Adams TT et al.; Several varieties of heat-sterilized broiler litter with 60% (wet basis, wb) moisture content were substrate in solid-state fermentations to produce biocontrol agents . Litter varieties included litter produced by one flock of broilers from medicated and non-medicated controlled rations, and litter produced by two flocks and four flocks on a single application of bedding material from medicated commercial sources . Litter preparations were inoculated with monocultures of Bacillus thuringiensis serovar japonensis strain Buibui, a pathogen of Japanese beetle larvae (Popillia japonica), or Pseudomonas fluorescens 2-79 . B . thuringiensis did not grow in unextracted 1-flock litter nor in water extracted litter, but grew in methanol extracted litter to 5 x 10(10) cell forming units (CFU)/g litter (dry weight, dw) and a spore count of 1 x 10(10) CFU/g litter (dw) . B . thuringiensis also grew in unprocessed 2-flock and 4-flock litter, achieving cell counts of 3 x 10(9) and 1 x 10(9) CFU/g litter (dw), respectively, and spore counts of 1 x 10(9) CFU/g litter (dw) . P . fluorescens grew in medicated 1-flock litter with no extraction to a cell density greater than 4 x 10(11) CFU/g litter (dw) . Bioassays in soil containing over 0.5% (db) litter fermented with B . thuringiensis resulted in over 90% mortality in 21 days for first instars of Japanese beetle when compared to a control treatment using compost without fermented litter . The investigations demonstrate that bacterial biocontrol agents produced via solid substrate fermentations using broiler poultry litter have potential in biocontrol applications in the soil environment. Arch Biochem Biophys, 2002 Jan 15, 397(2), 179 - 83 Siderophore-mediated iron uptake in fluorescent Pseudomonas: characterization of the pyoverdine-receptor binding site of three cross-reacting pyoverdines; Meyer JM et al.; Two Pseudomonas fluorescens and one Pseudomonas aeruginosa strains, although producing structurally different pyoverdines, demonstrated highly efficient cross-reactions when tested for pyoverdine-mediated iron uptake . A ferripyoverdine receptor-deficient mutant of the P . aeruginosa strain was unable to use any of the three pyoverdines . Moreover, the three strains presented each a specific outer membrane siderophore-receptor pattern . Thus, the capacity of using heterologous pyoverdines was related not to the presence of supplementary specific ferripyoverdine receptors but to the existence within the respective pyoverdine-peptide chains of a common dipeptide motif which should act as the receptor-binding site for the three pyoverdines . Other pyoverdines sharing the same motif but at another position within the peptide chain were not efficient in iron transport, demonstrating the importance of the spatial position of the binding site . (c)2002 Elsevier Science. Appl Environ Microbiol, 2002 Feb, 68(2), 791 - 8 Use of a marker organism to model the spread of central nervous system tissue in cattle and the abattoir environment during commercial stunning and carcass dressing; Daly DJ et al.; Due to concerns about a link between variant Creutzfeldt-Jakob disease in humans and similar prion protein-induced disease in cattle, i.e., bovine spongiform encephalopathy (BSE), strict controls are in place to exclude BSE-positive animals and/or specified risk materials including bovine central nervous system (CNS) tissue from the human food chain . However, current slaughter practice, using captive bolt guns, may induce disruption of brain tissues and mobilize CNS tissues into the bovine circulatory system, leading to the dispersion of CNS tissues (including prion proteins) throughout the derived carcass . This project used a marker (antibiotic-resistant) strain of Pseudomonas fluorescens to model the effects of commercial captive bolt stunning procedures on the movement of mobilized CNS material within slaughtered animals and the abattoir environment . The marker organism, introduced by injection through the bolt entry aperture or directly using a cartridge-fired captive bolt, was detected in the slaughter environment immediately after stunning and in the abattoir environment at each subsequent stage of the slaughter-dressing process . The marker organism was also detected on the hands of operatives; on slaughter equipment; and in samples of blood, organs, and musculature of inoculated animals . There were no significant differences between the results obtained by the two inoculation methods (P < 0.05) . This study demonstrates that material present in, or introduced into, the CNS of cattle during commercial captive bolt stunning may become widely dispersed across the many animate and inanimate elements of the slaughter-dressing environment and within derived carcasses including meat entering the human food chain. Antonie Van Leeuwenhoek, 2001 Sep, 79(3-4), 327 - 36 Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHAO; Bull CT et al.; In Pseudomonasfluorescens strain CHAO, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere . Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P . syringae pv . syringae as well as that of P . fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0 . This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5 . Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium . Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease . Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA . Independent point and deletion mutations arose at different sites in the gacA gene . In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased . In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions. Cytometry, 2002 Feb 1, 47(2), 118 - 26 Detection of the plant pathogenic bacterium Xanthomonas campestris pv . Campestris in seed extracts of Brassica sp . Applying fluorescent antibodies and flow cytometry; Chitarra LG et al.; BACKGROUND: Xanthomonas campestris pv . campestris (Xcc) is a seed-transmitted plant pathogenic bacterium that causes black rot of crucifers . Seed lots and plants are screened for contamination with this pathogen using plating or serological assays . These methods, however, are time consuming and not very sensitive, respectively . Therefore, flow cytometry (FCM) was evaluated as a tool for the rapid detection and quantification of Xcc cells labeled with a mixture of specific fluorescein isothicyanate (FITC)-monoclonal antibodies (mAb) in pure culture, in mixed cultures of Xcc with either the common saprophyte Pseudomonas fluorescens (Psf) or a nonpathogenic X . campestris isolate (Xc), and in crude seed extracts . METHODS: The mAb 18G12, conjugated with FITC, was tested at dilutions of 1:50, 1:100, 1:200, and 1:400 . For mixed suspensions of Xcc and Psf, mAb 18G12 was used at a dilution of 1:100 . The combination of mAbs 18G12, 2F4, and 20H6, all conjugated with FITC, was used at a dilution of 1:100 for the detection and quantification of Xcc cells in mixed suspensions containing Xcc and Xc and in crude seed extracts . The analyses were performed with a Coulter EPICS XL-MCL flow cytometer, at low flow rate during 2 min . RESULTS: Using FCM, Xcc cells labeled with FITC-conjugated mAbs (18G12, 2F4, and 20H6) were detected and quantified rapidly at low numbers, i.e., 10(3) colony-forming units per milliliter in pure and in mixed cultures with Psf . The presence of the nonpathogenic Xc in the seed extracts did not interfere with the FCM results . Xcc cells were distinguished from the cells of other organisms and from small particles present in the seed extract based on the high-intensity fluorescence of the labeled cells . CONCLUSION: The application of FCM in combination with FITC-conjugated mAbs appears to be a promising technique for the detection and quantification of Xcc cells in seed extracts of crucifers . J Bacteriol, 2002 Feb, 184(4), 1046 - 56 Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0; Heeb S et al.; In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA . This protein was purified from P . fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ . This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end . Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth . This signal appeared to be unrelated to N-acyl-homoserine lactones . A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression . Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease) . Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal . Overexpression of rsmA had a similar, albeit stronger negative effect . These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal . By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs. Mol Cells, 2001 Dec 31, 12(3), 347 - 52 Molecular cloning and characterization of Mycobacterium bovis BCG pcp gene encoding pyrrolidone carboxyl peptidase; Kim JK et al.; The Mycobacterium bovis bacilli Calmette-Guerin (BCG) pcp gene that encodes the pyrrolidone carboxyl peptidase (Pcp) was cloned from a lambdagtll genomic library and sequenced . The nucleotide sequence contains a 669 bp open reading frame coding for a protein of 222 amino acid residues with a calculated molecular mass of 23,209 Da . The deduced amino acid sequence is highly homologous to the Pcps from Bacillus amyloliquefaciens, Pseudomonas fluorescens, Bacillus subtilis, Streptococcus pyogenes, and Staphylococcus aureus . A multiple sequence alignment revealed highly conserved domains . The BCG pcp gene was overexpressed in Escherichia coli . The Pcp was purified to homogeneity . The recombinant protein was further confirmed by an enzymatic assay. Biochemistry, 2002 Jan 22, 41(3), 991 - 1000 Enzymatic hydrolysis of p-nitroacetanilide: mechanistic studies of the aryl acylamidase from Pseudomonas fluorescens; Stein RL; Aryl acylamidase (EC 3.1.5.13; AAA) catalyzes the hydrolysis of p-nitroacetanilide (PNAA) via the standard three-step mechanism of serine hydrolases: binding of substrate (K(s)), acylation of active-site serine (k(acyl)), and hydrolytic deacylation (k(deacyl)) . Key mechanistic findings that emerged from this study include that (1) AAA requires a deprotonated base with a pK(a) of 8.3 for expression of full activity toward PNAA . Limiting values of kinetic parameters at high pH are k(c) = 7 s(-1), K(m) = 20 microM, and k(c)/K(m) = 340 000 M(-1) s(-1) . (2) At pH 10, where all the isotope effects were conducted, k(c) is equally rate-limited by k(acyl) and k(deacyl) . (3) The following isotope effects were determined: (D)()2(O)(k(c)/K(m)) = 1.7 +/- 0.2, (D)()2(O)k(c) = 3.5 +/- 0.3, and (beta)(D)(k(c)/K(m)) = 0.83 +/- 0.04, (beta)(D)k(c) = 0.96 +/- 0.01 . These values, together with proton inventories for k(c)/K(m) and k(c), suggest the following mechanism: (i) The initial binding of substrate to enzyme to form the Michaelis complex is accompanied by solvation changes that generate solvent deuterium isotope effects originating from hydrogen ion fractionation at multiple sites on the enzyme surface . (ii) From within the Michaelis complex, the active site serine attacks the carbonyl carbon of PNAA with general-base catalysis to form a substantially tetrahedral transition state enroute to the acyl-enzyme . (iii) Finally, deacylation occurs through a process involving a rate-limiting solvent isotope effect, generating conformational change of the acyl-enzyme that positions the carbonyl bond in a polarizing environment that is optimal for attack by water. Mikrobiol Z, 2001 May-Jun, 63(3), 65 - 70 {Strains of Pseudomonas fluorescens 3 and Arthrobacter sp . 2--degradation of polycyclic aromatic hydrocarbons}; Soroka IaM et al.; Pseudomonas fluorescences 3 and Arthrobacter sp . 2 strains were isolated from the association of microorganisms--destructors of oil hydrocarbons and were selected for their ability to grow on media with phenanthrene as the only source of carbon and energy . The P . fluorescens 3 strain is able to grow on naphthalene, fluorene, phenanthrene, and anthracene . Arthrobacter sp . 2 strain did not grow on naphthalene, but was able to destruct phenanthrene and fluorene . The destruction activity of these strains both in pure and mixed culture towards the latter compounds has been studied . The both strains destructed phenanthrene added into the medium in the amount of 0.2 g/l, and phenanthrene destruction by Pseudomonas fluorescences 3 achieved 98.5% in 6 days and that by Arthrobacter sp . 93.5% in 23 days . Bacterial growth has been evaluated while measuring protein concentration in samples . Bacteria were inoculated in quantities equivalent to 0.0015-0.0020 mg protein/l . The cell protein concentration achieved 35-40 mg/l for Pseudomonas fluorescences 3, and 85-92 mg/l for Arthrobacter sp . by the end of incubation. J Ind Microbiol Biotechnol, 2001 Dec, 27(6), 337 - 42 Survival, root colonisation and biocontrol capacities of Pseudomonas fluorescens F113 LacZY in dry alginate microbeads; Russo A et al.; Cells of Pseudomonas fluorescens F113 LacZY were encapsulated in alginate and their survival and ability to colonise sugar beet were evaluated . To assess survival, the formulation, composed of dry alginate microbeads of 300- to 700-microm diameter, was stored 1 year at 28+/-2 and 4+/-2 degrees C and then tested against pathogenic fungi Pythium ultimum and Rhizoctonia solani in in vitro inhibition experiments . The same material was also used as inoculant for protection of sugar beet against Py . ultimum in microcosm experiments . The results obtained indicated that, although drying alginate beads resulted in a significant reduction of bacterial viability, the use of microbeads enabled a satisfactory level of root colonisation and protection, at least under microcosm conditions . The capability of the encapsulated cells to produce the antifungal metabolite 2,4-diacetylphloroglucinol (Phl) was not significantly affected by 12 months storage. Appl Environ Microbiol, 2002 Jan, 68(1), 65 - 72 Characterization of Pseudomonas spp . associated with spoilage of gilt-head sea bream stored under various conditions; Tryfinopoulou P et al.; The population dynamics of pseudomonads in gilt-head sea bream Mediterranean fish (Sparus aurata) stored under different conditions were studied . Phenotypic analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins were performed to identify a total of 106 Pseudomonas strains isolated from S . aurata stored under different temperatures (at 0, 10, and 20 degrees C) and packaging conditions (air and a modified atmosphere of 40% CO(2)-30% N(2)-30% O(2)) . Pseudomonas lundensis was the predominant species, followed by Pseudomonas fluorescens, while Pseudomonas fragi and Pseudomonas putida were detected less frequently . Fluorescent Pseudomonas strains dominated under air conditions, while proteolytic and less lipolytic strains dominated under modified-atmosphere packaging . Different storage conditions appear to govern the selection of pseudomonads in gilt-head sea bream fish. Microbiol Res, 2001, 156(4), 343 - 51 Motility and chemotactic response of Pseudomonas fluorescens toward chemoattractants present in the exudate of Macrophomina phaseolina; Singh T et al.; Pseudomonas fluorescens strains (LAM1-hydrophilic) and (LAM2-hydrophobic) showed positive chemotaxis towards attractants (sugars, amino acids, polyols and organic acids) present in the exudate of Macrophomina phaseolina (a soil-borne plant pathogenic fungus) . The varied response of motility traits such as speed, rate of change in direction (RCDI) and net to gross displacement ratio (NGDR) was observed for different chemoattractants . Swimming speed of the strains was highest in 10-fold diluted exudate or 100-1000 microM strength of different attractants, but further dilutions significantly decreased the swimming speed (P = 0.05) . Chemotactic response of P fluorescens was positively correlated with swimming speed (P = 0.05; r = 0.76) . Relative to control, the RCDI values decreased 1.5-fold in amino acids or sugars, and 1.2-fold in polyols or organic acids . With increase in swimming speed, the NGDR of both strains also increased, but the RCDI decreased . Both hydrophilic and hydrophobic strains did not show significant differences in their motility traits . The results demonstrate that M . phaseolina exudate contains chemical attractants that serve as signal for flagellar motility of P . fluorescens . Motile P fluorescens strains thus may consume fungal exudate as nutrients, and thus spores could offer a niche for these bacteria in soil. Phytochemistry, 2001 Dec, 58(8), 1297 - 303 Isolation and identification of N-mercapto-4-formylcarbostyril, an antibiotic produced by Pseudomonas fluorescens; Fakhouri W et al.; Pseudomonas fluorescens strain G308 isolated from barley leaves produces a novel antibiotic substance that was purified by preparative TLC and HPLC and identified as N-mercapto-4-formylcarbostyril (Cbs) by LC/DAD, IR, LC-ES(+)/MS, LC-ES(-)/MS, GC-EI/MS, LC-HRES(+)/MS, mass isotope ratios analysis, 1H NMR and 13C NMR analysis . The purified new antibiotic compound is effective against many phytopathogenic fungi in vitro . The compound inhibited at 25 ppm spore germination and germ tube growth of the following fungi; Fusarium oxysporum f . sp . lycopersici, Fusarium culmorum, Cladosporium cucumerinum and Colletotrichum lagenarium . At concentrations up to 125 ppm, the compound did not interfere with release of zoospores from sporangia and germination of encysted zoospores of Phytophthora infestans. Planta Med, 2001 Nov, 67(8), 732 - 6 Isolation from Cussonia barteri of 1'-O-chlorogenoylchlorogenic acid and 1'-O-chlorogenoylneochlorogenic acid, a new type of quinic acid esters; Papajewski S et al.; 1'-O-Chlorogenoylchlorogenic acid and 1'-O-chlorogenoylneochlorogenic acid, a new type of quinic acid esters, have been isolated, in addition to six known quinic acid esters, rutin, and a mixture of saponins, from the methanol extract of Cussonia barteri Seemann (Araliaceae) leaves collected in Cameroon . Structure determination was achieved by NMR, mass, IR, and UV spectroscopy . All compounds were tested for inhibitory activity on 5-lipoxygenase and cyclooxygenase-1, for antimicrobial activity against Bacillus subtilis, Pseudomonas fluorescens, and Cladosporium cucumerinum, and for haemolytic activity. FEMS Microbiol Lett, 2001 Nov 27, 205(1), 57 - 63 Impact of mutations in hemA and hemH genes on pyoverdine production by Pseudomonas fluorescens ATCC17400; Baysse C et al.; A Pseudomonas fluorescens Tn5 mutant, with decreased production of the siderophore pyoverdine, was obtained, with the transposon inserted in the hemA gene coding for glutamyl tRNA reductase, the enzyme that catalyzes the first step of heme biosynthesis . Since this mutant was leaky, a second round of transposition was needed to obtain a second mutant completely auxotrophic for the heme precursor delta-aminolevulinate (ALA) . Pyoverdine production by this mutant is ALA-dependent at concentrations above those needed to sustain growth . A transposon mutant in the hemH gene that encodes the enzyme ferrochelatase showing a characteristic red fluorescence upon UV exposure as a result of porphyrins accumulation, was obtained by selecting transconjugants on LB medium containing hemin . The DeltahemH mutant was characterized and the corresponding hemH gene sequenced . Antibodies against P . fluorescens HemH detected the protein both in soluble and membrane fractions of the wild-type and confirmed the absence of the enzyme in the mutant . The DeltahemH mutant failed to produce pyoverdine, but the production of the siderophore was restored by introduction of the Pseudomonas aeruginosa hemH gene in trans . These results indicate that de novo heme biosynthesis is needed for a normal level of siderophore pyoverdine production. Appl Environ Microbiol, 2001 Dec, 67(12), 5683 - 93 The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0; Schnider-Keel U et al.; A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases . This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and sigma(22)) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0 . The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae . Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction . Mucoidy strictly depended on the global regulator GacA . An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were . Expression of an algU'-'lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant . Overexpression of algU did not enhance tolerance towards osmotic stress . AlgU was found to be essential for tolerance of P . fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil . The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5% . In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P . fluorescens towards oxidative and heat stress . In conclusion, AlgU is a crucial determinant in the adaptation of P . fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment. Appl Environ Microbiol, 2001 Dec, 67(12), 5506 - 11 Homologous expression of the lipase and ABC transporter gene cluster, tliDEFA, enhances lipase secretion in Pseudomonas spp; Ahn JH et al.; The ABC transporter TliDEF was found to be an efficient secretory apparatus for extracellular lipase TliA in Pseudomonas fluorescens . For the enhanced secretion of the lipase, we tried to coexpress tliA and tliDEF in various Pseudomonas species . Whereas the coexpression of tliA and tliDEF was required for the lipase secretion in P . fragi, the expression of tliA was sufficient for the lipase secretion in P . fluorescens, P . syringae, and P . putida, indicating the existence of compatible ABC transporter in these species . However, P . fluorescens harboring tliDEFA secreted much more lipase than P . fluorescens harboring only tliA, but the tliDEF was functional only at temperatures below 30 degrees C . The recombinant P . fluorescens overexpressing tliDEFA showed the highest secretion level, 217 U/ml . OD (optical density) (28 microg/ml . OD) of lipase in Luria-Bertani medium under microaerated conditions . With the increase of aeration, the lipase production was decreased and the lipase seemed to be degraded as the cells entered the cell death phase . These results demonstrate that P . fluorescens can be used as a host system for the secretory production of the lipase using the ABC transporter, thus producing lipase in over 14% of the total protein. J Appl Microbiol, 2001 Nov, 91(5), 822 - 32 Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28; Meylheuc T et al.; AIMS: The influence of biosurfactant compounds produced by a strain of Pseudomonas fluorescens on the adhesion of Listeria monocytogenes LO28 to polytetrafluoroethylene (PTFE) and AISI 304 stainless steel surfaces was investigated . METHODS AND RESULTS: The biosurfactant was produced according to a simple, novel technique based on cultivation on nutrient agar . Adhesion studies were performed using L . monocytogenes cells cultured at 20 or 37 degrees C . CONCLUSIONS: A substrate-dependent behaviour of the LO28 strain (larger number of cells adhering to stainless steel than to PTFE), and a significant reduction (< 90%) in microbial adhesion levels through the prior adsorption of biosurfactants on stainless steel surfaces, which can be related to a change in the electron-donor characteristics of this substratum, was demonstrated . SIGNIFICANCE AND IMPACT OF THE STUDY: The prior adsorption of biosurfactants on solid surfaces may constitute a new and effective means of combating the implantation of pathogenic micro-organisms in food processing plants. Can J Microbiol, 2001 Oct, 47(10), 916 - 24 Evaluation of bacteria isolated from rice for plant growth promotion and biological control of seedling disease of rice; Adhikari TB et al.; Of 102 rhizoplane and endophytic bacteria isolated from rice roots and stems in California, 37% significantly (P < or = 0.05) inhibited the growth in vitro of two pathogens, Achlya klebsiana and Pythium spinosum, causing seedling disease of rice . Four endophytic strains were highly effective against seedling disease in growth pouch assays, and these were identified as Pseudomonas fluorescens (S3), Pseudomonas tolaasii (S20), Pseudomonas veronii (S21), and Sphingomonas trueperi (S12) by sequencing of amplified 16S rRNA genes . Strains S12, S20, and S21 contained the nitrogen fixation gene, nifD, but only S12 was able to reduce acetylene in pure culture . The four strains significantly enhanced plant growth in the absence of pathogens, as evidenced by increases in plant height and dry weight of inoculated rice seedlings relative to noninoculated rice . Three bacterial strains (S3, S20, and S21) were evaluated in pot bioassays and reduced disease incidence by 50%-73% . Strain S3 was as effective at suppressing disease at the lowest inoculum density (106 CFU/mL) as at higher density (10(8) CFU/mL or undiluted suspension) . This study indicates that selected endophytic bacterial strains have potential for control of seedling disease of rice and for plant growth promotion. J Microbiol Methods, 2001 Dec, 47(3), 315 - 22 A model that uses the induction phase of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 to quantify cell density in translucent porous media; Uesugi SL et al.; A cooled charge-coupled device (CCD) camera was used to follow the kinetics of induction of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 held either in aqueous suspensions minus sand, saturated or unsaturated translucent sand (0.348 and 0.07 cm(3) H(2)O/cm(3) of sand, respectively), and at cell densities ranging between 1 x 10(6) and 8.5 x 10(8) cells/ml . Before O(2) availability became a limiting factor, the rate of light emission (L) increased with the square of time (t) and linearly with increasing cell density (c) . A nonlinear model was developed that contains a "rate of increase in light emission" constant, B', which is determined directly from the slope of a plot of radical L/c against t . The model predicted the behavior of lux induction in HK44 under a variety of conditions . Similar B' values were determined {49.0-57.6 x 10(-10) light units/(cell min(2))} for cell suspensions held in aqueous medium minus sand, in saturated or unsaturated 40/50 grade sand (0.36 mm grain diameter) and in two other textural classes of translucent sand . Although both the growth phase, and the presence of glucose during lux induction affected the first detectable time (FDT) of bioluminescence by HK44 in sand, the kinetics of induction of light emission were similar among treatments (stationary phase cells plus glucose, B'=61.6+/-3.2, log phase cells plus glucose, B'=63.2+/-7.2) . The potential exists to use a combination of a CCD camera system, an inducible lux gene containing bioluminescent bacterium, and a light transmission chamber to nonintrusively visualize and quantify in real time the interactions between bacterial growth and unsaturated flow of water and solutes in porous media. Biomacromolecules, 2000 Fall, 1(3), 493 - 500 Enzyme-catalyzed ring-opening copolymerization of 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (MBC) with trimethylene carbonate (TMC): synthesis and characterization; al-Azemi TF et al.; Enzymatic ring-opening copolymerization of 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (MBC) with trimethylene carbonate (TMC) was investigated . A route to aliphatic polycarbonates decorated with pendent carboxylic acid groups is demonstrated . Lipase from Pseudomonas fluorescens (AK) was selected to perform the copolymerization at various monomers feed ratios . Copolymers with different composition were prepared by varying the monomer feed ratio from 10% to 80% MBC . 1H, 13C, and 1H-13C HETCOR NMR spectra were used to analyze the microstructure of the copolymers . The 1H NMR spectra indicated lower incorporation of TMC in the copolymer than was expected from the monomer feed ratio . Analysis of the 13C spectra did not indicate an ordered structure but instead suggested the formation of random polymers . This was further confirmed by the thermal data obtained for representative samples . The thermal properties at different feed ratios of poly{MBC-co-TMC} copolymers were investigated by differential scanning calorimetry (DSC) . No melting temperature (Tm) for either homopolymers or copolymers was observed . A plot of 1/Tg(K) vs the weight composition of MBC in the copolymers was constructed and was consistent with copolymers that tend toward random distribution; this was confirmed from the NMR data. J Inorg Biochem, 2001 Nov, 87(1-2), 1 - 8 Modulation of TCA cycle enzymes and aluminum stress in Pseudomonas fluorescens; Hamel RD et al.; Oxalic acid plays a pivotal role in the adaptation of the soil microbe Pseudomonas fluorescens to aluminum (Al) stress . Its production via the oxidation of glyoxylate necessitates a major reconfiguration of the enzymatic reactions involved in the tricarboxylic acid (TCA) cycle . The demand for glyoxylate, the precursor of oxalic acid appears to enhance the activity of isocitrate lyase (ICL) . The activity of ICL, an enzyme that participates in the cleavage of isocitrate to glyoxylate and succinate incurred a 4-fold increase in the Al-stressed cells . However, the activity of isocitrate dehydrogenase, a competitor for the substrate isocitrate, appeared to be diminished in cells exposed to Al compared to the control cells . While the demand for oxalate in Al-stressed cells also negatively influenced the activity of the enzyme alpha-ketoglutarate dehydrogenase complex, no apparent change in the activity of malate synthase was recorded . Thus, it appears that the TCA cycle is tailored in order to generate the necessary precursor for oxalate synthesis as a consequence of Al-stress. Gene, 2001 Oct 31, 278(1-2), 107 - 14 Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene cluster of Pseudomonas fluorescens; Morea A et al.; The organization of the alginate gene cluster in Pseudomonas fluorescens was characterized . A bank of genomic DNA from P . fluorescens was mobilized to a strain of Pseudomonas aeruginosa with a transposon insertion (al