J Biotechnol, 1999 Sep 24, 75(1), 71 - 75 Development of a cold resistant mutant of plant growth promoting Pseudomonas fluorescens and its functional characterization; Mishra M et al.; A cold resistant mutant of plant growth promoting Pseudomonas fluorescens GRS(1), was isolated following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment . The mutant was able to grow and promote root and shoot elongation of wheat both at 25 and 10 degrees C, a temperature at which the wild type was unable to proliferate and function . In an effort to determine the mechanistic basis for this behavior, it was observed that following growth at 10 degrees C, the mutant synthesized some new proteins . SDS-polyacrylamide gel electrophoresis of the protein synthesized by the mutant revealed the presence of one major protein with a molecular mass of 13.5 kDa . However, at this point the function of this protein in not known. J Microbiol Methods, 2000 Apr, 40(2), 135 - 45 A comparison of enumeration methods for culturable Pseudomonas fluorescens cells marked with green fluorescent protein; Cassidy MB et al.; The detection of bacteria in environmental samples using genetic markers is valuable in microbial ecology . The green fluorescent protein (GFP) reporter gene was studied under nutrient starvation conditions at 4 degrees C, 23 degrees C and 30 degrees C in Pseudomonas fluorescens R2fG1 cells tagged with a red-shifted gfp . Fluorescence intensity was not significantly different in cells maintained in a buffer for at least 48 days at all the tested temperatures . gfp-Tagged R2fG1 cells were introduced into bulk soil microcosms and soil microcosms with wheat seedlings . GFP-marked cells were enumerated immediately after inoculation into soil and again in soil and root samples after 10 days . Counts of culturable colonies were obtained from drop plates using 5-microl aliquots of serial dilutions viewed with an epifluorescent microscope . Traditional spread plates (using 100-microl aliquots) and the most-probable-number (MPN) method using a spectrofluorometer were also used to enumerate the GFP-marked Pseudomonas cells in soil, rhizosphere and rhizoplane samples . Microcolonies were visualized on root surfaces under the epifluorescent microscope after immobilizing in agar and incubation for 24 h . Counts from traditional spread plates were significantly higher (P<0.05) than the population estimates of the MPN method for all treatments at any sampling time . Counts using the drop plate method, however, were not significantly different (P<0.05) except in one treatment, and provided similar estimates in half the time of spread plates and at an estimated third of the cost. Can J Microbiol, 2000 Jan, 46(1), 28 - 37 Influence of growth and environmental conditions on cell surface hydrophobicity of Pseudomonas fluorescens in non-specific adhesion; Jana TK et al.; The relative cell surface hydrophobicity (CSH) of 18 soil isolates of Pseudomonas fluorescens, determined by phase exclusion, hydrophobic interaction chromatography (HIC), electrostatic interaction chromatography (ESIC), and contact angle, revealed large degrees of variability . Variation in the adhesion efficiency to Macrophomina phaseolina of the hyphae/sclerotia of these isolates was also examined . Two such isolates with maximum (32.8%; isolate 12-94) and minimum (12%; isolate 30-94) CSH were selected for further study . Early- to mid-log exponential cells of these isolates were more hydrophobic than those in stationary phase, and the CSH of these isolates was also influenced by fluctuations in temperatures and pH . Isolate 12-94 exhibited high CSH (32.3%) at 30 degrees C, compared to lower values (28-24%) in the higher temperature range (35-40 degrees C) . Increasing concentrations of either Zn2+, Fe3+, K+, and Mg2+ in the growth medium were associated with the increased CSH . Trypsin, pepsin, and proteinase K (75 to 150 micrograms.mL-1) reduced the CSH of isolate 12-94 cells . CSH was reduced, following exposure to DTT, SDS, Triton X-100, or Tween 80 . Prolonged exposure of cells to starvation (60 days) also caused a significant decline in CSH . Several protein bands (18, 21, 23, 26 kDa) of the outer cell membrane were absent in 60-day starved cells compared to unstarved cells . In conclusion, our findings demonstrate that CSH of P . fluorescens isolates may contribute to nonspecific attachment/adhesion onto M . phaseolina hyphae/sclerotia, and the efficiency of adhesion is regulated by growth and other environmental conditions. J Food Prot, 2000 Feb, 63(2), 162 - 6 Survival of Pseudomonas fluorescens and Salmonella typhimurium after electron beam and gamma irradiation of refrigerated beef; Chung MS et al.; The radurization effects of gamma ray and electron beam irradiation at 1.5 and 3.0 kGy on beef steaks inoculated with Salmonella Typhimurium and Pseudomonas fluorescens were investigated during 8 days of storage at 5 degrees C . Total bacterial counts and numbers of Salmonella Typhimurium and P . fluorescens were analyzed at 2-day intervals . Total bacterial counts of samples irradiated by both gamma rays and electron beam were significantly (P < 0.05) reduced by 3.8 to 5.3 log CFU/g . Salmonella Typhimurium was not detectable during the experimental period . P . fluorescens counts of beef samples irradiated by gamma rays at both 1.5 and 3.0 kGy were not detected; however, P . fluorescens in samples irradiated by electron beam at 1.5 and 3.0 kGy was recovered after 2 days, and bacterial counts reached 7.8 and 6.9 log CFU/g, respectively . Both gamma ray and electron beam irradiation reduced total bacterial counts initially, possibly extending shelf life . Irradiation was very effective in destroying Salmonella Typhimurium; however, P . fluorescens was not completely eliminated by electron beam irradiation . Consequently, gamma ray irradiation was more effective than electron beam irradiation in the destruction of P . fluorescens. Chemotherapy, 2000 Mar-Apr, 46(2), 129 - 34 Effects of antibiotics on adherence of Pseudomonas aeruginosa and Pseudomonas fluorescens to A549 pneumocyte cells; Di Martino P et al.; The aim of this study was to evaluate the effect of antibiotics at subminimal inhibitory concentrations (sub-MIC) on fluorescent pseudomonas adherence to A549 pneumocyte cells . Pseudomonas fluorescens MF0 isolated from contaminated raw milk and Pseudomonas aeruginosa NK125502 isolated from a cystic fibrosis patient's lung adhered to A549 cells . As previously shown for P . aeruginosa, P . fluorescens bound to A549 cells in a dose-dependent manner over a wide range of bacterial concentrations . Bacterial growth in the presence of polymyxin B or gentamicin at MIC/2 had no effect on the adherence of NK125502 and MF0 to A549 cells . Instead, MIC/2 and MIC/8 of cefsulodin or chloramphenicol decreased the adherence of the two strains . A decrease in MF0 adherence was also observed with cefsulodin at MIC/32 . We conclude that, in addition to their antibacterial activity, cefsulodin and chloramphenicol could be effective in preventing Pseudomonas adherence to respiratory epithelium . J Bacteriol, 2000 Mar, 182(5), 1215 - 25 Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin; Schnider-Keel U et al.; The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens . A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined . Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor . The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes . In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase . Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium . 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth . Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG . In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added . The phlF mutant was insensitive to 2,4-DAPG addition . A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription . Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium . In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production . PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host . In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites . This mechanism, which depends on phlF function, may help P . fluorescens to produce homeostatically balanced amounts of extracellular metabolites. Lett Appl Microbiol, 1999 Nov, 29(5), 298 - 302 ppGpp accumulation in Pseudomonas aeruginosa and Pseudomonas fluorescens subjected to nutrient limitation and biocide exposure; Greenway DL et al.; The effect of a commonly used biocide, 1,2-benzisothiazolin-3-one (BIT) on ppGpp accumulation in the pathogen, Pseudomonas aeruginosa PAO1, and an environmental isolate, Ps . fluorescens, was examined . It is concluded that BIT is able to induce a stringent response in Ps . aeruginosa and Ps . fluorescens, determined by the rapid accumulation of ppGpp following addition of BIT to exponentially-growing cells . Western blot analysis of whole-cell extracts with anti-RelA antibody demonstrated that both species contain a RelA homologue . This is the first report of a RelA-like protein in pseudomonads. Appl Environ Microbiol, 2000 Feb, 66(2), 763 - 8 An improved spectrophotometric method to study the transport, attachment, and breakthrough of bacteria through porous media; Deshpande PA et al.; This study reports an improved spectrophotometric method for studying bacterial (Pseudomonas fluorescens UPER-1) transport and attachment in saturated porous media (silica sand) . While studying the effect of ionic strength by the traditional packed-column spectrophotometric method, we encountered an artifact . The absorbance of a well-stirred bacterial suspension was found to decrease with time in the presence of high concentrations of sodium and potassium phosphate salts (> or = 10(-2) M) as the cells continued to age in a resting stage . Our results show that collision efficiency and a bed ripening index will be in error by as much as 20% if breakthrough is measured by the traditional spectrophotometric technique . We present an improved experimental technique that will minimize the artifact and should substantially advance the understanding of bacteria transport in porous media. Appl Environ Microbiol, 2000 Feb, 66(2), 487 - 92 Quinolobactin, a new siderophore of Pseudomonas fluorescens ATCC 17400, the production of which is repressed by the cognate pyoverdine; Mossialos D et al.; Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin) . The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type . The mutant was also characterized by substantially increased uptake of (59)Fe-quinolobactin . The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin . Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture . Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine. Biochemistry, 2000 Feb 8, 39(5), 985 - 91 Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands; Ponyi T et al.; Aromatic amino acids are believed to play a pivotal role in carbohydrate-binding proteins, by forming hydrophobic stacking interactions with the sugar rings of their target ligands . Family 10 cellulose-binding modules (CBM10s), present in a number of cellulases and xylanases expressed by Pseudomonas fluorescens subsp . cellulosa, contain two tyrosine and three tryptophan residues which are highly conserved . To investigate whether these amino acids play an important role in the interaction of CBM10 from P . fluorescens subsp . cellulosa xylanase A (Pf Xyn10A) with cellulose, each of these residues was changed to alanine in CBM10 expressed as a discrete module or fused to the catalytic domain of Pf Xyn10A (CBM10-CD), and the capacity of the mutant proteins of CBM10-CD to bind the polysaccharide was evaluated . The data showed that W22A, W24A, and Y8A bound very weakly to cellulose compared to the wild-type protein, while Y12A retained its capacity to interact with the glucose polymer . When the W7A mutation was introduced into CBM10 the protein domain did not accumulate in Escherichia coli . In contrast, the W7A mutant of CBM10-CD was efficiently expressed in E . coli, although the protein bound very weakly to cellulose . NMR spectra of wild-type CBM10, W22A, and W24A were very similar, suggesting that the mutations did not significantly affect the protein fold . Titration of wild-type CBM10, W22A, and W24A with N-bromosuccinimide indicated that Trp22 and Trp24 were on the surface of the protein, while Trp7 was buried . Collectively, these data indicate that Trp22, Trp24, and Tyr8 play a direct role in the binding of Pf Xyn10A CBM10 to cellulose . The results are discussed in the light of the three-dimensional structure of CBM10 {Raghothama, S., Simpson, P . J., Szabo, L., Nagy, T., Gilbert, H . J., and Williamson, M . P . (2000) Biochemistry 39, 978-984}. Biochemistry, 2000 Feb 8, 39(5), 978 - 84 Solution structure of the CBM10 cellulose binding module from Pseudomonas xylanase A; Raghothama S et al.; Plant cell wall hydrolases generally have a modular structure consisting of a catalytic domain linked to one or more noncatalytic carbohydrate-binding modules (CBMs), whose common function is to attach the enzyme to the polymeric substrate . Xylanase A from Pseudomonas fluorescens subsp . cellulosa (Pf Xyn10A) consists of a family 10 catalytic domain, an N-terminal family IIa cellulose-binding module, and an internal family 10 cellulose-binding module . The structure of the 45-residue family 10 CBM has been determined in solution using NMR . It consists of two antiparallel beta-sheets, one with two strands and one with three, with a short alpha-helix across one face of the three-stranded sheet . There is a high density of aromatic residues on one side of the protein, including three aromatic residues (Tyr8, Trp22, and Trp24), which are exposed and form a flat surface on one face, in a classical polysaccharide-binding arrangement . The fold is closely similar to that of the oligonucleotide/oligosaccharide-binding (OB) fold, but appears to have arisen by convergent evolution, because there is no sequence similarity, and the presumed binding sites are on different faces. Appl Environ Microbiol, 2000 Jan, 66(1), 369 - 74 Role of leaf surface sugars in colonization of plants by bacterial epiphytes; Mercier J et al.; The relationship between nutrients leached onto the leaf surface and the colonization of plants by bacteria was studied by measuring both the abundance of simple sugars and the growth of Pseudomonas fluorescens on individual bean leaves . Data obtained in this study indicate that the population size of epiphytic bacteria on plants under environmentally favorable conditions is limited by the abundance of carbon sources on the leaf surface . Sugars were depleted during the course of bacterial colonization of the leaf surface . However, about 20% of readily utilizable sugar, such as glucose, present initially remained on fully colonized leaves . The amounts of sugars on a population of apparently identical individual bean leaves before and after microbial colonization exhibited a similar right-hand-skewed distribution and varied by about 25-fold from leaf to leaf . Total bacterial population sizes on inoculated leaves under conditions favorable for bacterial growth also varied by about 29-fold and exhibited a right-hand-skewed distribution . The amounts of sugars on leaves of different plant species were directly correlated with the maximum bacterial population sizes that could be attained on those species . The capacity of bacteria to deplete leaf surface sugars varied greatly among plant species . Plants capable of supporting high bacterial population sizes were proportionally more depleted of leaf surface nutrients than plants with low epiphytic populations . Even in species with a high epiphytic bacterial population, a substantial amount of sugar remained after bacterial colonization . It is hypothesized that residual sugars on colonized leaves may not be physically accessible to the bacteria due to limitations in wettability and/or diffusion of nutrients across the leaf surface. J Inorg Biochem, 1999 Aug 30, 76(2), 99 - 104 Oxalic acid production and aluminum tolerance in Pseudomonas fluorescens; Hamel R et al.; 13C NMR studies on intact cells from Al-stressed Pseudomonas fluorescens incubated with citric acid or Al-citrate yielded peaks at 158 and 166 ppm that were attributable to free and complexed oxalic acid, respectively . The presence of oxalic acid was further confirmed with the aid of oxalate oxidase . These peaks were not discernable in experiments performed with cells taken from control cultures . Enzymatic analyses of cell fractions showed the highest production of oxalic acid in the inner membrane fraction of Al-stressed cells incubated with glyoxylate . There was an eight-fold increase in the synthesis of oxalic acid in the inner membrane fraction from the Al-stressed cells compared to the control cells . Although oxalic acid production was observed when citrate, Al-citrate and isocitrate were utilized as substrates, the inner membrane fraction did not mediate the formation of oxalic acid from glycine/pyruvate, glycolic acid, oxaloacetate or ascorbate . These data suggest that the increased oxalic acid production in response to Al stress is effected via the oxidation of glyoxylate. Plant Mol Biol, 1999 Nov, 41(4), 537 - 49 Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge; van Wees SC et al.; Selected strains of nonpathogenic rhizobacteria from the genus Pseudomonas are capable of eliciting broad-spectrum induced systemic resistance (ISR) in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR) . In Arabidopsis, the ISR pathway functions independently of salicylic acid (SA) but requires responsiveness to jasmonate and ethylene . Here, we demonstrate that known defense-related genes, i.e . the SA-responsive genes PR-1, PR-2, and PR-5, the ethylene-inducible gene Hel, the ethylene- and jasmonate-responsive genes ChiB and Pdf1.2, and the jasmonate-inducible genes Atvsp, Lox1, Lox2, Pall, and Pin2, are neither induced locally in the roots nor systemically in the leaves upon induction of ISR by Pseudomonas fluorescens WCS417r . In contrast, plants infected with the virulent leaf pathogen Pseudomonas syringae pv . tomato (Pst) or expressing SAR induced by preinfecting lower leaves with the avirulent pathogen Pst(avrRpt2) exhibit elevated expression levels of most of the defense-related genes studied . Upon challenge inoculation with Pst, PR gene transcripts accumulated to a higher level in SAR-expressing plants than in control-treated and ISR-expressing plants, indicating that SAR involves potentiation of SA-responsive PR gene expression . In contrast, pathogen challenge of ISR-expressing plants led to an enhanced level of Atvsp transcript accumulation . The otherjasmonate-responsive defense-related genes studied were not potentiated during ISR, indicating that ISR is associated with the potentiation of specific jasmonate-responsive genes. Lipids, 1999 Nov, 34(11), 1159 - 66 Wax ester-synthesizing activity of lipases; Tsujita T et al.; The synthesis/hydrolysis of wax esters was studied in an aqueous solution using purified rat pancreatic lipase, porcine pancreatic carboxylester lipase, and Pseudomonas fluorescens lipase . The equilibrium between wax ester synthesis and hydrolysis favored ester formation at neutral pH . The synthesizing activities were measured using free fatty acid or triacylglycerol as the acyl donor and an equimolar amount of long-chain alcohol as the acyl acceptor . When oleic acid and hexadecanol emulsified with gum arabic were incubated with these lipases, wax ester was synthesized, in a dose- and time-dependent manner, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was about 0.9/0.1 . These lipases catalyzed the hydrolysis of palmityl oleate emulsified with gum arabic, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was also about 0.9/0.1 . The apparent equilibrium ratio of wax ester/free fatty acid catalyzed by lipase depended on incubation pH and fatty alcohol chain length . When equimolar amounts of trioleoylglycerol and fatty acyl alcohol were incubated with pancreatic lipase, carboxylester lipase, or P . fluorescens lipase, wax esters were synthesized dose-dependently . These results suggest that lipases can catalyze the synthesis of wax esters from free fatty acids or through degradation of triacylglycerol in an aqueous medium. J Bacteriol, 1999 Dec, 181(24), 7545 - 51 Cloning and characterization of the Pseudomonas fluorescens ATP-binding cassette exporter, HasDEF, for the heme acquisition protein HasA; Idei A et al.; Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluorescens no . 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA . The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system . P . fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marcescens . The P . fluorescens Has exporter secreted HasA proteins from P . fluorescens and P . aeruginosa but not S . marcescens HasA in Escherichia coli, whereas the Has exporter from S . marcescens allowed secretion of all three HasA proteins . The P . fluorescens HasDEF system also promoted the secretion of the lipase and alkaline protease of P . fluorescens . Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates . Chimeric HasA proteins containing both P . fluorescens and S . marcescens sequences were produced and tested for secretion through the Has exporters . The C-terminal region of HasA was shown to be involved in the secretion specificity of the P . fluorescens Has exporter. J Microbiol Methods, 1999 Nov, 38(3), 177 - 82 An automated technique for most-probable-number (MPN) analysis of densities of phagotrophic protists with lux AB labelled bacteria as growth medium; Ekelund F et al.; An automated modification of the most-probable-number (MPN) technique has been developed for enumeration of phagotrophic protozoa . The method is based on detection of prey depletion in micro titre plates rather than on presence of protozoa . A transconjugant Pseudomonas fluorescens DR54 labelled with a luxAB gene cassette was constructed, and used as growth medium for the protozoa in the micro titre plates . The transconjugant produced high amounts of luciferase which was stable and allowed detection for at least 8 weeks . Dilution series of protozoan cultures and soil suspensions were inoculated into micro titre plates amended with a suspension of the transconjugant . After 45 days measurement of light emission allowed detection of individual wells in the titre plates, where protozoan grazing had removed the inoculated bacteria. J Appl Microbiol, 1999 Sep, 87(3), 454 - 63 Ecological basis for biocontrol of damping-off disease by pseudomonas fluorescens 54/96 Ellis RJ, Timms-Wilson TM, Beringer JE, Rhodes D, Renwick A, Stevenson L, Bailey MJ. Pseudomonas fluorescens 54/96, originally isolated from the rhizosphere of sugar beet, has been shown to be commercially effective in field trials for the suppression of a number of fungal diseases of seedlings . In vitro and microcosm-based assays revealed that both the timing and method of application of bacteria were important for effective control of Pythium ultimum, the causative agent of damping-off disease . Following transposon mutagenesis (Tn5lac), mutants deficient for the suppression of Pythium ultimum infections of peas were isolated . Three major classes of insertional mutants of Ps . fluorescens 54/96 were identified which either inhibited sporulation, reduced mycelial growth or affected the regulation of bacterial metabolic activity . Evaluation of the metabolic capability of pathogen and antagonist revealed evidence for direct competition, as both the fungus and bacterium had similar sole carbon source nutrient utilization profiles . Further comparisons of the activity of the transposon mutants indicated that although the mechanisms of disease control were multifactorial, the most significant factor was the prevention of rapid spore germination in the presence of pea seeds. Arch Microbiol, 1999 Dec, 172(6), 354 - 63 Identification and molecular characterization of the eugenol hydroxylase genes (ehyA/ehyB) of Pseudomonas sp . strain HR199; Priefert H et al.; The gene loci ehyA and ehyB, which are involved in the bioconversion of eugenol to coniferyl alcohol by Pseudomonas sp . strain HR199 (DSM 7063), were identified as the structural genes of a eugenol hydroxylase that represents an enzyme of the flavocytochrome c class . These genes were localized downstream of the eugenol catabolism genes vanA and vanB, encoding vanillate-O-demethylase, on an EcoRI fragment (E230) that has recently been cloned from a Pseudomonas sp . strain HR199 genomic library . The gene encoding the cytochrome c subunit (ehyA) was identified on a subfragment (K18) of E230 by complementation of a nitrosoguanidine-induced, eugenol-negative mutant of strain HR199 . The nucleotide sequences of fragment K18 and adjacent regions were determined, revealing open reading frames of 354 and 1,554 bp that represent ehyA and ehyB, respectively . These genes are most probably organized in one operon together with a third open reading frame (ORF2) of 687 bp that was located between ehyA and ehyB . The deduced amino acid sequences of ehyA and ehyB exhibited up to 29 and 55% amino acid identity to the corresponding subunits of p-cresol methylhydroxylase from Pseudomonas putida . Moreover, the amino-terminal sequences of the alpha- and beta-subunits reported recently for a eugenol dehydrogenase of Pseudomonas fluorescens E118 corresponded well with appropriate regions of ehyA and ehyB . The sequence of ORF2 and the deduced amino acid sequence exhibited no significant similarities to any DNA or amino acid sequence from the databases . The eugenol hydroxylase genes were amplified by PCR, cloned in pBluescript SK(-), and functionally expressed in Escherichia coli . Transfer of a DNA fragment comprising ehyA and ehyB to various strains of Pseudomonas species that were unable to utilize eugenol as a carbon source conferred to these bacteria the ability to grow on this substrate. J Agric Food Chem, 1999 Oct, 47(10), 4093 - 9 Effect of freezing and microbial growth on myoglobin derivatives of beef; Ben Abdallah M et al.; The effect of freezing and bacterial growth on the discoloration of beef was assessed by measuring myoglobin derivatives myoglobin (MB), oxymyoglobin (MBO(2)), and metmyoglobin (METMB) on the surfaces of fresh and frozen-thawed packaged beef cuts stored at 2 degrees C and analyzed after 0, 3, 6, 9, and 12 days of storage . MB, MBO(2), and METMB concentrations were measured spectrophotometrically . Frozen-thawed beef samples experienced less "blooming" (conversion of MB to MBO(2)) and more rapid discoloration than fresh cuts during storage . By day 3, >20% METMB was formed in the frozen-thawed samples, whereas the fresh samples reached this value after day 6 of storage . The rates of MB oxidation were similar (P > 0.05) for sterile and frozen-thawed inoculated (Pseudomonas fluorescens at a rate of 1.5 colony forming units/cm(2).cm(2) area) samples from day 0 through day 6 of storage . For storage periods of less than a week, bacterial growth is not a major cause of meat discoloration . After day 6, the high bacterial growth rate resulted in a rapid increase in METMB formation . Possible mechanisms for MB oxidation in frozen-thawed beef are suggested. Bioorg Med Chem, 1999 Oct, 7(10), 2169 - 73 Directed evolution of an esterase: screening of enzyme libraries based on pH-indicators and a growth assay; Bornscheuer UT et al.; In order to resolve a sterically hindered 3-hydroxy ethyl ester, which was not accepted as substrate by 20 wild-type hydrolases, a directed evolution of an esterase from Pseudomonas fluorescens (PFE) was performed . Mutations were introduced using the mutator strain Epicurian coli XL1-Red . Enzyme libraries derived from seven mutation cycles were assayed on minimal media agar plates supplemented with pH indicators (neutral red and crystal violet), thus allowing the identification of active esterase variants by the formation of a red color caused by a pH decrease due to the released acid . A further selection criteria was introduced by using the corresponding glycerol estar, because release of the carbon source glycerol facilitates growth on minimal media . By this strategy, one double mutant (A209D and L181V) of PFE was identified, which hydrolyzed the 3-hydroxy ethyl ester in a stereoselective manner (25% ee for the remaining ester, E approximate to 5). Res Microbiol, 1999 Sep, 150(7), 447 - 56 Genetic studies of a thermoregulated gene in the psychrotrophic bacterium Pseudomonas fluorescens; Regeard C et al.; In the psychrotrophic bacterium Pseudomonas fluorescens, some genes are thermoregulated: they are maximally expressed at a particular temperature within the broad range of temperatures that allow growth of this bacterium . To study this regulation, random transcriptional insertion fusions were obtained by means of mini-Tn5lacZ1 or mini-Tn5luxAB transposition . One fusion was studied in which beta-galactosidase production was maximal at a low-growth temperature . The mutated gene (that we call xsf) was highly homologous to xseA from Escherichia coli (and from other bacteria) which encodes the large subunit of exonuclease VII . Genetic tools were constructed in order to analyse and manipulate this fusion: a plasmid derived from R68.45 was used for chromosome transfer and a replacement vector was constructed to allow in situ marker exchange of the mini-Tn5lacZ1 by an Hg(r) interposon . This vector was used to make double mutants and hence to study the effect of the insertion in xsf on the expression of other fusions . Six genes were thereby identified with a decreased expression in an xsf- background and with different characteristics of thermoregulation. Ecotoxicol Environ Saf, 1999 Oct, 44(2), 154 - 9 Mobility of the organochlorine compound dicofol in soil promoted by Pseudomonas fluorescens; Brunninger BM et al.; The genetic modified Pseudomonas fluorescens Br 12, resistant to kanamycin and rifampycin, was used to follow the cotransport of the organochlorine acaricide dicofol through a nonsterilized soil column . P . fluorescens was found to bioaccumulate dicofol with the highest bioconcentration factor of 279 within 30 min . Separate soil column experiments where applied P . fluorescens or {14C}dicofol were submitted to heavy rain simulation did not reveal any correlation between the distribution patterns of P . fluorescens and {14C}dicofol in the leachate fractions (r = 0.3) . Similar experiments with P . fluorescens that previously had bioaccumulated {14C}dicofol demonstrated a high correlation of these bacteria and radioactivity in the leachate fractions (r = 0.8) . The total recovery of radioactivity in the leachate, when {14C}dicofol was previously bioaccumulated in bacteria, was more than two times higher (4.5%) than the total recovery of radioactivity in the leachate when {14C} dicofol was directly applied in the soil (2%) . This indicates cotransport by Pseudomonas . Fractionation and analysis of soil columns indicated that most of the bioaccumulated dicofol was rapidly released and adsorbed in soil, while bacteria moved down by leaching. Proc Natl Acad Sci U S A, 1999 Nov 23, 96(24), 14073 - 8 Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites; Blumer C et al.; The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria . In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide . GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism . Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not . A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation . GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site . The gene coding for the global translational repressor RsmA of P . fluorescens was cloned . RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade . Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect . Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation. J Agric Food Chem, 1999 Apr, 47(4), 1681 - 6 Kinetics of thermal inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F: influence of pH, calcium, and protein; Schokker EP et al.; The influence of pH, calcium ion activity, protein, and enzyme purification on the kinetics of heat inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F was studied in the temperature range 80-120 degrees C . At pH 5.5-8.6 the rate of inactivation increased slightly with increasing pH values . The pH dependence of inactivation suggests that the inactivation mechanism is mainly through deamidation . Calcium ion activity had no influence on the kinetics of heat inactivation of the proteinase . Addition of 1.8% sodium caseinate to the enzyme solution slightly decreased the heat stability of the proteinase, possibly because part of the inactivation of the proteinase is caused by aggregation to casein . Purification of the proteinase did not change the rate of thermal inactivation. J Agric Food Chem, 1999 Feb, 47(2), 648 - 54 SPME-MS-MVA as an electronic nose for the study of off-flavors in milk; Marsili RT; A new technique using solid-phase microextraction, mass spectrometry, and multivariate analysis (SPME-MS-MVA) was developed for the study of off-flavors in milk . The analytical column of a GC/MS system was replaced with a 1-m deactivated fused-silica column, which served as a transfer line to deliver volatiles extracted from milk samples with a Carboxen-SPME fiber to the mass spectrometer . Mass fragmentation data resulting from the unresolved milk volatile components were subjected to MVA . Principal component analysis based on SPME-MS-MVA provided rapid differentiation of control reduced-fat milk (2% butterfat content) samples from reduced-fat milk samples abused by light, heat, copper, and microbial contamination . The three psychrotrophic bacteria studied included Pseudomonas fluorescens, Pseudomonas aureofaciens, and Pseudomonas putrefaciens . SPME-MS-MVA is rapid and offers significant advantages over commercial electronic nose instruments currently being used as quality assurance tools to differentiate normal-tasting food and beverage samples from those containing off-flavors and malodors. J Agric Food Chem, 1999 Jan, 47(1), 318 - 21 Synthesis and biological activity of enantiomeric pairs of phosphosulfonate herbicides; Spangler LA et al.; The phosphosulfonates are a new class of soil-active herbicides which control a variety of annual grass and broadleaf weeds . Chirality at the phosphorus atom afforded the opportunity to explore stereospecific requirements for herbicidal activity . Chiral (hydroxymethyl)phosphinate intermediates were enzymatically resolved (Pseudomonas fluorescens lipase) from the racemic mixtures and then used to prepare two pairs of enantiomeric phosphosulfonates . Biological testing of the enantiomeric phosphosulfonate herbicides demonstrated that, in each case, the herbicidal activity was attributed to the (+) enantiomer and that the (+) enantiomer is more active than the racemate. Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1559 - 72 Pseudomonas gessardii sp . nov . and Pseudomonas migulae sp . nov., two new species isolated from natural mineral waters; Verhille S et al.; Twenty-five non-identified fluorescent Pseudomonas strains isolated from natural mineral waters were previously clustered into three phenotypic subclusters, XIIIb, XVa and XVc . These strains were characterized genotypically in the present study . DNA-DNA hybridization results and DNA base composition analysis revealed that these strains were members of two new species, for which the names Pseudomonas gessardii sp . nov . (type strain CIP 105469T) and Pseudomonas migulae sp . nov . (type strain CIP 105470T) are proposed . P . gessardii included 13 strains from phenotypic subclusters XVa and XVc . P . migulae included 10 strains from phenotypic subcluster XIIIb . The levels of DNA-DNA relatedness ranged from 71 to 100% for P . gessardii and from 74 to 100% for P . migulae . The G + C content of the DNA of each type strain was 58 mol% . DNA similarity levels, measured with 67 reference strains of Pseudomonas species, were below 55%, with delta Tm values of 13 degrees C or more . The two new species presented basic morphological characteristics common to all pseudomonads . Various phenotypic features were found to differentiate them: P . gessardii strains utilized L-arabitol, myo-inositol, adonitol, xylitol and meso-erythritol as carbon sources, whereas P . migulae strains assimilated L-arabinose, D-xylose, D-saccharate, meso-tartrate, tricarballylate, D-glucuronate, D-galacturonate, phenylacetate and histamine . The complete 16S rRNA sequences of each type strain were determined and compared with those of the type strains of Pseudomonas species . Finally, a phylogenetic tree was inferred from sequence analysis and demonstrated that the two new species fell into the 'Pseudomonas fluorescens intrageneric cluster' . To date, their clinical significance is unknown. Appl Environ Microbiol, 1999 Nov, 65(11), 4837 - 47 Biochemical and genetic analyses of ferulic acid catabolism in Pseudomonas sp . Strain HR199; Overhage J et al.; The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding beta-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol in Pseudomonas sp . strain HR199 (DSM7063), were localized on a DNA region covered by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp . strain HR199 genomic library in the cosmid pVK100 . The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes . To confirm the function of the structural genes fcs and ech, they were cloned and expressed in Escherichia coli . Recombinant strains harboring both genes were able to transform ferulic acid to vanillin . The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the fcs and ech gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis . To prove the essential involvement of the fcs, ech, and aat genes in the catabolism of ferulic acid and eugenol in Pseudomonas sp . strain HR199, these genes were inactivated separately by the insertion of omega elements . The corresponding mutants Pseudomonas sp . strain HRfcsOmegaGm and Pseudomonas sp . strain HRechOmegaKm were not able to grow on ferulic acid or on eugenol, whereas the mutant Pseudomonas sp . strain HRaatOmegaKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type . In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed . The aat gene product was shown not to be involved in this catabolism, thus excluding a beta-oxidation analogous degradation pathway for ferulic acid . Moreover, the function of the ech gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomonas sp . strain HR199 proceeds via a similar pathway to that recently described for Pseudomonas fluorescens AN103. J Dairy Sci, 1999 Oct, 82(10), 2078 - 83 Autolysis of the proteinase from Pseudomonas fluorescens; Kumura H et al.; The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C . A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found . Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues . The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE . Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme . Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline . Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-terminal amino acid sequence analysis in an effort to determine the cleavage sites . The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites. FEMS Microbiol Ecol, 1999 Nov 1, 30(3), 217 - 227 Chitinolytic activity of Pseudomonas fluorescens isolates from barley and sugar beet rhizosphere; Neiendam Nielsen M et al.; Twelve isolates of Pseudomonas fluorescens, isolated from barley and sugar beet rhizosphere as antagonists towards the plant pathogenic microfungi Rhizoctonia solani and Pythium ultimum, showed chitinolytic activity in batch cultures when grown in media without exogenous chitin . Enzyme tests in cultures demonstrated a complete array of chitinolytic enzymes . Endochitinase and chitobiosidase activities seemed to be tightly coupled in the isolates, while there was no relationship to N-acetyl-glucosaminidase activity . Endochitinase activity, showing hydrolysis of chitin polymers, was found to be extracellular in all isolates, although the most active isolates also retained a cell-bound fraction . Isoelectric focusing gel electrophoresis of supernatants containing extracellular enzyme activity showed that all isolates produced one native endochitinase in logarithmic phase . This enzyme was subsequently modified into several isozymes by extracellular processing as the cultures aged in stationary phase . The 12 isolates could be grouped into seven isozymic patterns . Detailed studies of three selected isolates showed that extracellular release of endochitinase activity also took place in stationary phase . The results, however, indicated that in stationary phase regulation of both the overall production of native enzyme and the subsequent formation of isozymes were different among the P . fluorescens isolates. Biochim Biophys Acta, 1999 Sep 3, 1446(3), 377 - 82 The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no . 33; Kawai E et al.; In Pseudomonas fluorescens no . 33, the lipase gene is clustered with the genes for alkaline protease, AprDEF exporter, and two homologue proteins of Serratia serine proteases (pspA and pspB) . Secretion of the lipase and alkaline protease through AprDEF was shown in the Escherichia coli cells . Interestingly, the E . coli cells carrying the pspA gene secreted PspA to the media AprDEF-independently. FEMS Microbiol Lett, 1999 Oct 15, 179(2), 501 - 6 A mutant of gluconobacter oxydans deficient in gluconic acid dehydrogenase Gupta A, Felder M, Verma V V, Cullum J, Qazi GN. Gluconobacter oxydans ATCC 9937 was subjected to transposon mutagenesis using Tn5 . A non-pigmented mutant was shown to be defective in gluconic acid dehydrogenase and to produce gluconic acid from glucose, whereas the parent strain produced 2, 5-diketogluconic acid . Cloning and sequencing of the region containing the Tn5 insertion showed that the insertion point occurred in an open reading frame homologous (42% amino acid identity) to the ribF genes of Pseudomonas fluorescens and Escherichia coli . The resulting lack of a riboflavin cofactor would explain the loss of enzyme activity. Mol Plant Microbe Interact, 1999 Oct, 12(10), 911 - 8 Identification of a locus in arabidopsis controlling both the expression of rhizobacteria-mediated induced systemic resistance (ISR) and basal resistance against Pseudomonas syringae pv . tomato; Ton J et al.; Selected nonpathogenic rhizobacteria with biological disease control activity are able to elicit an induced systemic resistance (ISR) response that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR) . Ten ecotypes of Arabidopsis thaliana were screened for their potential to express rhizobacteria-mediated ISR and pathogen-induced SAR against the leaf pathogen Pseudomonas syringae pv . tomato DC3000 (Pst) . All ecotypes expressed SAR . However, of the 10 ecotypes tested, ecotypes RLD and Wassilewskija (Ws) did not develop ISR after treatment of the roots with nonpathogenic Pseudomonas fluorescens WCS417r bacteria . This nonresponsive phenotype was associated with relatively high susceptibility to Pst infection . The F1 progeny of crosses between the non-responsive ecotypes RLD and Ws on the one hand, and the responsive ecotypes Columbia (Col) and Landsberg erecta (Ler) on the other hand, were fully capable of expressing ISR and exhibited a relatively high level of basal resistance, similar to that of their WCS417r-responsive parent . This indicates that the potential to express ISR and the relatively high level of basal resistance against Pst are both inherited as dominant traits . Analysis of the F2 and F3 progeny of a Col x RLD cross revealed that inducibility of ISR and relatively high basal resistance against Pst cosegregate in a 3:1 fashion, suggesting that both resistance mechanisms are monogenically determined and genetically linked . Neither the responsiveness to WCS417r nor the relatively high level of basal resistance against Pst were complemented in the F1 progeny of crosses between RLD and Ws, indicating that RLD and Ws are both affected in the same locus, necessary for the expression of ISR and basal resistance against Pst . The corresponding locus, designated ISR1, was mapped between markers B4 and GL1 on chromosome 3 . The observed association between ISR and basal resistance against Pst suggests that rhizobacteria-mediated ISR against Pst in Arabidopsis requires the presence of a single dominant gene that functions in the basal resistance response against Pst infection. Appl Environ Microbiol, 1999 Oct, 65(10), 4646 - 51 Green fluorescent protein-marked Pseudomonas fluorescens: localization, viability, and activity in the natural barley rhizosphere; Normander B et al.; The gfp-tagged Pseudomonas fluorescens biocontrol strain DR54-BN14 was introduced into the barley rhizosphere . Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3- to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid . Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity . At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere . Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period . No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent . In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability. Appl Environ Microbiol, 1999 Oct, 65(10), 4320 - 8 Nitrogen availability to Pseudomonas fluorescens DF57 is limited during decomposition of barley straw in bulk soil and in the barley rhizosphere; Jensen LE et al.; The availability of nitrogen to Pseudomonas fluorescens DF57 during straw degradation in bulk soil and in barley rhizosphere was studied by introducing a bioluminescent reporter strain (DF57-N3), responding to nitrogen limitation, to model systems of varying complexity . DF57-N3 was apparently not nitrogen limited in the natural and sterilized bulk soil used for these experiments . The soil was subsequently amended with barley straw, representing a plant residue with a high carbon-to-nitrogen ratio (between 60 and 100) . In these systems the DF57-N3 population gradually developed a nitrogen limitation response during the first week of straw decomposition, but exclusively in the presence of the indigenous microbial population . This probably reflects the restricted ability of DF57 to degrade plant polymers by hydrolytic enzymes . The impact of the indigenous population on nitrogen availability to DF57-N3 was mimicked by the cellulolytic organism Trichoderma harzianum Rifai strain T3 when coinoculated with DF57-N3 in sterilized, straw-amended soil . Limitation occurred concomitantly with fungal cellulase production, pointing to the significance of hydrolytic activity for the mobilization of straw carbon sources, thereby increasing the nitrogen demand . Enhanced survival of DF57-N3 in natural soil after straw amendment further indicated that DF57 was cross-fed with carbon/energy sources . The natural barley rhizosphere was experienced by DF57-N3 as an environment with restricted nitrogen availability regardless of straw amendment . In the rhizosphere of plants grown in sterilized soil, nitrogen limitation was less severe, pointing to competition with indigenous microorganisms as an important determinant of the nitrogen status for DF57-N3 in this environment . Hence, these studies have demonstrated that nitrogen availability and gene expression in Pseudomonas is intimately linked to the structure and function of the microbial community . Further, it was demonstrated that the activities of cellulolytic microorganisms may affect the availability of energy and specific nutrients to a group of organisms deficient in hydrolytic enzyme activities. FEMS Microbiol Lett, 1999 Sep 15, 178(2), 327 - 35 Cloning, sequence analysis, and expression of ansB from Pseudomonas fluorescens, encoding periplasmic glutaminase/asparaginase; Huser A et al.; A gene (ansB) encoding a class II glutaminase/asparaginase has been cloned from Pseudomonas fluorescens and characterized by DNA sequencing, promoter analysis and heterologous expression in Escherichia coli . We show that ansB is monocistronic and depends on the alternate sigma factor sigma 54 for expression . A second open reading frame located downstream of ansB is highly homologous to a number of bacterial genes that encode secreted endonucleases of unknown function. Mikrobiologiia, 1999 May-Jun, 68(3), 330 - 9 {Characteristics of lipopolysaccharide from Pseudomonas fluorescens (biovar I)}; Zdorovenko GM et al.; Lipopolysaccharide (LPS) of the Pseudomonas fluorescens strain IMV 7769 (biovar I) was isolated and investigated . Fractions of the structural parts of the LPS macromolecule, lipid A, the core oligosaccharide, and the O-specific polysaccharide (O-PS), were obtained in a homogeneous state . 2-Hydroxydecanoic, 3-hydroxydecanoic, dodecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, hexadecanoic, octadecanoic, hexadecenoic, and octadecenoic fatty acids were identified in lipid A . In the hydrophilic moiety of lipid A, after acid hydrolysis, several amino acids, phosphoethanolamine, glucosamine, and three unidentified peaks forming a separate cluster together with glucosamine were found . Lipid A was shown to be phosphorylated . Glucose, fucose, rhamnose, glucosamine, galactosamine, two unidentified amino sugars, 2-keto-3-deoxyoctulonic acid (KDO), heptose, ethanolamine, phosphoethanolamine, and alanine were identified in the core oligosaccharide . O-PS of the LPS consisted of repeating trisaccharide fragments that included residues of amino sugars: 4-acetamido-4,6-dideoxy-D-galactose, 2-acetamido-2,6-dideoxy-D-glucose, and 2-acetamido-2,6-dideoxy-L-glucose . During growth, the strain under study excreted exocellular LPS (ELPS) into the medium . The LPS studied was similar to the LPS of the earlier investigated strains P . fluorescens (biovar I) IMV 1152 and IMV 1433 in the structure of O-PS, but differed from them in the composition of both lipid A and the core oligosaccharide . The LPS of the strain studied differed from LPS of the type strain P . fluorescens IMV 4125 (ATCC 13525) in all characteristics determined. Biol Chem, 1999 Jul-Aug, 380(7-8), 1029 - 33 Directed evolution of an esterase from Pseudomonas fluorescens . Random mutagenesis by error-prone PCR or a mutator strain and identification of mutants showing enhanced enantioselectivity by a resorufin-based fluorescence assay; Henke E et al.; The gene encoding an esterase from Pseudomonas fluorescens (PFE) was subjected to random mutagenesis by error-prone PCR or by using the mutator strain Epicurian coli XL1-Red . Enzyme libraries were then created in microtiter plates by expression of PFE-variants in E . coli . These were assayed for improved enantioselectivity in a Beckman robot system using optically pure (R)- or (S)-3-phenylbutyric acid resorufin esters, resulting in the identification of several mutants showing up to almost two-fold enantioselectivity (E(true) = 5.2 to 6.6) compared to wild-type PFE (E(true) = 3.5). Biochem J, 1999 Oct 1, 343 Pt 1, 215 - 24 A modular cinnamoyl ester hydrolase from the anaerobic fungus Piromyces equi acts synergistically with xylanase and is part of a multiprotein cellulose-binding cellulase-hemicellulase complex; Fillingham IJ et al.; A collection of clones, isolated from a Piromyces equi cDNA expression library by immunoscreening with antibodies raised against affinity purified multienzyme fungal cellulase-hemicellulase complex, included one which expressed cinnamoyl ester hydrolase activity . The P . equi cinnamoyl ester hydrolase gene (estA) comprised an open reading frame of 1608 nt encoding a protein (EstA) of 536 amino acids and 55540 Da . EstA was modular in structure and comprised three distinct domains . The N-terminal domain was closely similar to a highly conserved non-catalytic 40-residue docking domain which is prevalent in cellulases and hemicellulases from three species of anaerobic fungi and binds to a putative scaffolding protein during assembly of the fungal cellulase complex . The second domain was also not required for esterase activity and appeared to be an atypically large linker comprising multiple tandem repeats of a 13-residue motif . The C-terminal 270 residues of EstA contained an esterase catalytic domain that exhibited overall homology with a small family of esterases, including acetylxylan esterase D (XYLD) from Pseudomonas fluorescens subsp . cellulosa and acetylxylan esterase from Aspergillus niger . This region also contained several smaller blocks of residues that displayed homology with domains tentatively identified as containing the essential catalytic residues of a larger group of serine hydrolases . A truncated variant of EstA, comprising the catalytic domain alone (EstA'), was expressed in Escherichia coli as a thioredoxin fusion protein and was purified to homogeneity . EstA' was active against synthetic and plant cell-wall-derived substrates, showed a marked preference for cleaving 1-->5 ester linkages between ferulic acid and arabinose in feruloylated arabino-xylo-oligosaccharides and was inhibited by the serine-specific protease inhibitor aminoethylbenzene-sulphonylfluoride . EstA' acted synergistically with xylanase to release more than 60% of the esterified ferulic acid from the arabinoxylan component of plant cell walls . Western analysis confirmed that EstA is produced by P . equi and is a component of the aggregated multienzyme cellulase-hemicellulase complex . Hybrid proteins, harbouring one, two or three iterations of the conserved 40-residue fungal docking domain fused to the reporter protein glutathione S-transferase, were produced . Western blot analysis of immobilized P . equi cellulase-hemicellulase complex demonstrated that each of the hybrid proteins bound to a 97 kDa polypeptide in the extracellular complex. J Mol Biol, 1999 Sep 10, 292(1), 87 - 96 Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase; Eppink MH et al.; p-Hydroxybenzoate hydroxylase (PHBH) is the archetype of the family of NAD(P)H-dependent flavoprotein aromatic hydroxylases . These enzymes share a conserved FAD-binding domain but lack a recognizable fold for binding the pyridine nucleotide . We have switched the coenzyme specificity of strictly NADPH-dependent PHBH from Pseudomonas fluorescens by site-directed mutagenesis . To that end, we altered the solvent exposed helix H2 region (residues 33-40) of the FAD-binding domain . Non-conservative selective replacements of Arg33 and Tyr38 weakened the binding of NADPH without disturbing the protein architecture . Introduction of a basic residue at position 34 increased the NADPH binding strength . Double (M2) and quadruple (M4) substitutions in the N-terminal part of helix H2 did not change the coenzyme specificity . By extending the replacements towards residues 38 and 40, M5 and M6 mutants were generated which were catalytically more efficient with NADH than with NADPH . It is concluded that specificity in P . fluorescens PHBH is conferred by interactions of Arg33, Tyr38 and Arg42 with the 2'-phosphate moiety of bound NADPH, and that introduction of an acidic group at position 38 potentially enables the recognition of the 2'-hydroxy group of NADH . This is the first report on the coenzyme reversion of a flavoprotein aromatic hydroxylase . Bioorg Med Chem, 1999 Aug, 7(8), 1497 - 503 Stereospecificity of Pseudomonas fluorescens kynureninase for diastereomers of beta-methylkynurenine; Cyr LV et al.; The diastereomers of beta-methyl-L-kynurenine were prepared by preparative ozonolysis of the respective diastereomers of beta-methyl-L-tryptophan . A practical method for preparative enzymatic resolution of the diastereomers of beta-methyltryptophan was developed using carboxypeptidase A digestion of the N-trifluoroacetyl derivatives . The stereochemical assignment was confirmed by X-ray crystal structure determination of (2S, 3R)-threo-beta-methyl-L-tryptophan . (2S,3S)-erythro-beta-Methyl-L-kynurenine is a slow substrate for kynureninase from Pseudomonas fluorescens (k(cat)/K(m) = 0.1% that of L-kynurenine), producing anthranilic acid, while (2S,3R)-threo-L-kynurenine is about 390-fold less reactive than erythro . Rapid-scanning stopped-flow measurements show that beta-methyl substitution affects the rate of alpha-deprotonation of the L-kynurenine-pyridoxal-5'-phosphate Schiffs base . This is consistent with the stereoelectronic requirements of the reaction . These results are the first demonstration that beta-substituted kynurenines can be substrates for kynureninase, and may be useful in the design of mechanism-based inhibitors. Biosci Biotechnol Biochem, 1999 Jul, 63(7), 1165 - 70 Identification of a member of the serralysin family isolated from a psychrotrophic bacterium, Pseudomonas fluorescens 114; Kumeta H et al.; An extracellular metalloprotease named No . 114 protease is one of the major secretions of a psychrotrophic bacterium, Pseudomonas fluorescens 114, the cold-adaptation mechanism of which has not been identified . In this study, we purified and cloned No . 114 protease, which is a single polypeptide having a molecular mass of 47 kDa . This protease contains a zinc-binding motif (HEXXHXUGUXH: X, arbitrary amino acid; U, bulky hydrophobic amino acid), glycine-rich repeats (GGXGXD) and no cysteine residue, which are the features specifically found in serralysin subfamily . No . 114 protease has its maximum activity at the temperature of 35-40 degrees C, which is about 20 degrees C lower than that of a serralysin from a mesophilic bacterium, Pseudomonas aeruginosa . All these results imply that No . 114 protease from this psychrophilic bacterium is a unique member of the serralysin group characterized by a low optimal temperature. Mol Plant Microbe Interact, 1999 Aug, 12(8), 720 - 7 Systemic resistance in Arabidopsis induced by rhizobacteria requires ethylene-dependent signaling at the site of application; Knoester M et al.; Root colonization of Arabidopsis thaliana by the nonpathogenic, rhizosphere-colonizing, biocontrol bacterium Pseudomonas fluorescens WCS417r has been shown to elicit induced systemic resistance (ISR) against Pseudomonas syringae pv . tomato (Pst) . The ISR response differs from the pathogen-inducible systemic acquired resistance (SAR) response in that ISR is independent of salicylic acid and not associated with pathogenesis-related proteins . Several ethylene-response mutants were tested and showed essentially normal symptoms of Pst infection . ISR was abolished in the ethylene-insensitive mutant etr1-1, whereas SAR was unaffected . Similar results were obtained with the ethylene-insensitive mutants ein2 through ein7, indicating that the expression of ISR requires the complete signal-transduction pathway of ethylene known so far . The induction of ISR by WCS417r was not accompanied by increased ethylene production in roots or leaves, nor by increases in the expression of the genes encoding the ethylene biosynthetic enzymes 1-aminocyclopropane-1-carboxylic (ACC) synthase and ACC oxidase . The eir1 mutant, displaying ethylene insensitivity in the roots only, did not express ISR upon application of WCS417r to the roots, but did exhibit ISR when the inducing bacteria were infiltrated into the leaves . These results demonstrate that, for the induction of ISR, ethylene responsiveness is required at the site of application of inducing rhizobacteria. Appl Environ Microbiol, 1999 Sep, 65(9), 4085 - 93 Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil; Hojberg O et al.; The root-colonizing bacterium Pseudomonas fluorescens CHA0 was used to construct an oxygen-responsive biosensor . An anaerobically inducible promoter of Pseudomonas aeruginosa, which depends on the FNR (fumarate and nitrate reductase regulation)-like transcriptional regulator ANR (anaerobic regulation of arginine deiminase and nitrate reductase pathways), was fused to the structural lacZ gene of Escherichia coli . By inserting the reporter fusion into the chromosomal attTn7 site of P . fluorescens CHA0 by using a mini-Tn7 transposon, the reporter strain, CHA900, was obtained . Grown in glutamate-yeast extract medium in an oxystat at defined oxygen levels, the biosensor CHA900 responded to a decrease in oxygen concentration from 210 x 10(2) Pa to 2 x 10(2) Pa of O(2) by a nearly 100-fold increase in beta-galactosidase activity . Half-maximal induction of the reporter occurred at about 5 x 10(2) Pa . This dose response closely resembles that found for E . coli promoters which are activated by the FNR protein . In a carbon-free buffer or in bulk soil, the biosensor CHA900 still responded to a decrease in oxygen concentration, although here induction was about 10 times lower and the low oxygen response was gradually lost within 3 days . Introduced into a barley-soil microcosm, the biosensor could report decreasing oxygen concentrations in the rhizosphere for a 6-day period . When the water content in the microcosm was raised from 60% to 85% of field capacity, expression of the reporter gene was elevated about twofold above a basal level after 2 days of incubation, suggesting that a water content of 85% caused mild anoxia . Increased compaction of the soil was shown to have a faster and more dramatic effect on the expression of the oxygen reporter than soil water content alone, indicating that factors other than the water-filled pore space influenced the oxygen status of the soil . These experiments illustrate the utility of the biosensor for detecting low oxygen concentrations in the rhizosphere and other soil habitats. Appl Environ Microbiol, 1999 Sep, 65(9), 3828 - 33 Amplified fragment length polymorphism fingerprinting of Pseudomonas strains from a poultry processing plant; Geornaras I et al.; Molecular typing has been used previously to identify and trace dissemination of pathogenic and spoilage bacteria associated with food processing . Amplified fragment length polymorphism (AFLP) is a novel DNA fingerprinting technique which is considered highly reproducible and has high discriminatory power . This technique was used to fingerprint 88 Pseudomonas fluorescens and Pseudomonas putida strains that were previously isolated from plate counts of carcasses at six processing stages and various equipment surfaces and environmental sources of a poultry abattoir . Clustering of the AFLP patterns revealed a high level of diversity among the strains . Six clusters (clusters I through VI) were delineated at an arbitrary Dice coefficient level of 0.65; clusters III (31 strains) and IV (28 strains) were the largest clusters . More than one-half (52.3%) of the strains obtained from carcass samples, which may have represented the resident carcass population, grouped together in cluster III . By contrast, 43.2% of the strains from most of the equipment surfaces and environmental sources grouped together in cluster IV . In most cases, the clusters in which carcass strains from processing stages grouped corresponded to the clusters in which strains from the associated equipment surfaces and/or environmental sources were found . This provided evidence that there was cross-contamination between carcasses and the abattoir environment at the DNA level . The AFLP data also showed that strains were being disseminated from the beginning to the end of the poultry processing operation, since many strains associated with carcasses at the packaging stage were members of the same clusters as strains obtained from carcasses after the defeathering stage. Acta Microbiol Pol, 1999, 48(1), 73 - 8 Indigenous microflora responses to introduction of cyanogenic strains of Pseudomonas fluorescens into soil; Piotrowska-Seget Z et al.; The effects of cyanogenic Pseudomonas fluorescens strains introduced into soil on the kinetic of colony formation and bacterial community structure were investigated . About 7.8 x 10(8) and 1.2 x 10(9) cfu per g dry soil of TA1 and B2 were added to the soil portions, respectively . The parameters of colony formation by heterotrophic soil bacteria were determined . The bacterial community structure and phenotypic diversity were studied using concept of r/K strategies and echophysiological index, respectively . The physiological state of indigenous heterotrophic bacteria and gram-negative group did not change under the influence of the cyanogenic strains introduced . Phenotypic diversity of the soil bacteria also did not change significantly . However, some short-term shifts in community structure of indigenous heterotrophic bacteria were noticed . This study shows that the introduction of great numbers of cyanogenic P . fluorescens strains could be safely used as potential agents in biological control of soil-born pathogens. Structure Fold Des, 1999 Aug 15, 7(8), 977 - 88 Crystal structure of Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway; Serre L et al.; BACKGROUND: In plants and photosynthetic bacteria, the tyrosine degradation pathway is crucial because homogentisate, a tyrosine degradation product, is a precursor for the biosynthesis of photosynthetic pigments, such as quinones or tocophenols . Homogentisate biosynthesis includes a decarboxylation step, a dioxygenation and a rearrangement of the pyruvate sidechain . This complex reaction is carried out by a single enzyme, the 4-hydroxyphenylpyruvate dioxygenase (HPPD), a non-heme iron dependent enzyme that is active as a homotetramer in bacteria and as a homodimer in plants . Moreover, in humans, a HPPD deficiency is found to be related to tyrosinemia, a rare hereditary disorder of tyrosine catabolism . RESULTS: We report here the crystal structure of Pseudomonas fluorescens HPPD refined to 2.4 A resolution (Rfree 27.6%; R factor 21.9%) . The general topology of the protein comprises two barrel-shaped domains and is similar to the structures of Pseudomonas 2,3-dihydroxybiphenyl dioxygenase (DHBD) and Pseudomonas putida catechol 2,3-dioxygenase (MPC) . Each structural domain contains two repeated betaalpha betabeta betaalpha modules . There is one non-heme iron atom per monomer liganded to the sidechains of His161, His240, Glu322 and one acetate molecule . CONCLUSIONS: The analysis of the HPPD structure and its superposition with the structures of DHBD and MPC highlight some important differences in the active sites of these enzymes . These comparisons also suggest that the pyruvate part of the HPPD substrate (4-hydroxyphenylpyruvate) and the O2 molecule would occupy the three free coordination sites of the catalytic iron atom . This substrate-enzyme model will aid the design of new inhibitors of the homogentisate biosynthesis reaction. J Food Prot, 1997 Jan, 60(1), 23 - 7 Monoclonal antibodies and an indirect ELISA for detection of psychrotrophic bacteria in refrigerated milk; Gutierrez R et al.; Monoclonal antibodies generated against live cells of Pseudomonas fluorescens have been used in an indirect ELISA format for the detection of Pseudomonas spp . and related psychrotrophic bacteria in refrigerated milk . The immunorecognition of monoclonal antibodies adsorbed to bacteria bound to the wells of a microtiter plate was performed with rabbit anti-mouse immunoglobulins conjugated to horseradish peroxidase . Subsequent enzymic conversion of the substrate resulted in distinct absorbance differences when assaying milk samples containing psychrotrophic bacteria in the range 10(5) to 10(9) CFU ml(-1) . The detection threshold for the ELISA assay developed in this work is 10(5) CFU ml(-1). Biochem J, 1999 Sep 1, 342 ( Pt 2), 473 - 80 The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism; Gill J et al.; Xylanase A (Pf Xyn10A), in common with several other Pseudomonas fluorescens subsp . cellulosa polysaccharidases, consists of a Type II cellulose-binding domain (CBD), a catalytic domain (Pf Xyn10A(CD)) and an internal domain that exhibits homology to Type X CBDs . The Type X CBD of Pf Xyn10A, expressed as a discrete entity (CBD(X)) or fused to the catalytic domain (Pf Xyn10A'), bound to amorphous and bacterial microcrystalline cellulose with a K(a) of 2.5 x 10(5) M(-1) . CBD(X) exhibited no affinity for soluble forms of cellulose or cello-oligosaccharides, suggesting that the domain interacts with multiple cellulose chains in the insoluble forms of the polysaccharide . Pf Xyn10A' was 2-3 times more active against cellulose-hemicellulose complexes than Pf Xyn10A(CD); however, Pf Xyn10A' and Pf Xyn10A(CD) exhibited the same activity against soluble substrates . CBD(X) did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did not potentiate Pf Xyn10A(CD) when not covalently linked to the enzyme . There was no substantial difference in the affinity of full-length Pf Xyn10A and the enzyme's Type II CBD for cellulose . The activity of Pf Xyn10A against cellulose-hemicellulose complexes was similar to that of Pf Xyn10A', and a derivative of Pf Xyn10A in which the Type II CBD is linked to the Pf Xyn10A(CD) via a serine-rich linker sequence {Bolam, Cireula, McQueen-Mason, Simpson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J . 331, 775-781} . These data indicate that CBD(X) is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of catalysis, is evident between the Type II and X CBDs of the xylanase. Can J Microbiol, 1999 May, 45(5), 369 - 76 Cometabolic oxidation of phenanthrene to phenanthrene trans-9,10-dihydrodiol by Mycobacterium strain S1 growing on anthracene in the presence of phenanthrene; Tongpim S et al.; Mycobacterium strain S1, originally described as Rhodococcus strain S1 by chemotaxonomic criteria, was isolated by growth on anthracene, and is unable to use any of nine other polycyclic aromatic compounds as carbon source . Metabolism of phenanthrene during growth on anthracene as sole carbon source results in the accumulation of traces of a dihydrodiol metabolite in the growth medium, which, by comparison with authentic standards, has been tentatively identified as phenanthrene trans-9,10-dihydrodiol . Anthracene metabolites were ruled out on the basis of comparisons with authentic anthracene dihydrodiols from Pseudomonas fluorescens D1 and chemically synthesized anthrols . The original source of phenanthrene for dihydrodiol production was phenanthrene present as a < 1% contaminant in the anthracene used as carbon source . However, addition of further phenanthrene to the anthracene growth medium increased the level of phenanthrene trans-9,10-dihydrodiol formed . Mycobacterium strain S1 also produced phenanthrene trans-9,10-dihydrodiol when grown in a glucose-salts medium in the presence of phenanthrene . This dihydrodiol is a dead-end metabolite, and neither it nor its parent hydrocarbon are able to support the growth of Mycobacterium strain S1 . Studies with metyrapone and ancimidol, which did not inhibit growth on anthracene but did inhibit formation of phenanthrene trans-9,10-dihydrodiol, suggest it is likely the product of a cytochrome P450 monooxygenase-like activity. Biochemistry, 1999 Aug 10, 38(32), 10489 - 98 Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens; Slatner M et al.; To characterize catalysis by NAD-dependent long-chain mannitol 2-dehydrogenases (MDHs), the recombinant wild-type MDH from Pseudomonas fluorescens was overexpressed in Escherichia coli and purified . The enzyme is a functional monomer of 54 kDa, which does not contain Zn(2+) and has B-type stereospecificity with respect to hydride transfer from NADH . Analysis of initial velocity patterns together with product and substrate inhibition patterns and comparison of primary deuterium isotope effects on the apparent kinetic parameters, (D)k(cat), (D)(k(cat)/K(NADH)), and (D)(k(cat)/K(fructose)), show that MDH has an ordered kinetic mechanism at pH 8.2 in which NADH adds before D-fructose, and D-mannitol and NAD are released in that order . Isomerization of E-NAD to a form which interacts with D-mannitol nonproductively or dissociation of NAD from the binary complex after isomerization is the slowest step (>/=110 s(-)(1)) in D-fructose reduction at pH 8.2 . Release of NADH from E-NADH (32 s(-)(1)) is the major rate-limiting step in mannitol oxidation at this pH . At the pH optimum for D-fructose reduction (pH 7.0), the rate of hydride transfer contributes significantly to rate limitation of the catalytic cascade and the overall reaction . (D)(k(cat)/K(fructose)) decreases from 2.57 at pH 7.0 to a value of </=1 above pH 9.6, corresponding to the pK of 9.34 observed in the pH profile of k(cat)/K(fructose) . Therefore, hydride transfer is not pH-dependent, and D-fructose is not sticky at pH 7.0 . A comparison of the kinetic data of MDH and mammalian sorbitol dehydrogenase, presumably involved in detoxification metabolism, is used to point out a physiological function of MDH in the oxidation of D-mannitol with high specificity and fluxional efficiency under prevailing reaction conditions in vivo. J Bacteriol, 1999 Aug, 181(16), 4746 - 54 Influence of a putative ECF sigma factor on expression of the major outer membrane protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens; Brinkman FS et al.; The gene encoding OprF, a major outer membrane protein in Pseudomonas species (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene . We now report the identification of a novel putative ECF (extracytoplasmic function) sigma factor gene, sigX, located immediately upstream of oprF in both Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens OE 28.3 and show that disruption of this gene significantly reduces OprF expression . In P . aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronic oprF combined with a polar effect due to termination of a transcript containing sigX and oprF . Comparison of sigX-disrupted and wild-type cell transcripts by primer extension indicated that monocistronic transcription of oprF occurs from two overlapping promoters, one that is SigX-dependent and resembles ECF sigma factor promoters in its minus-35 region and another promoter that is independent of SigX and is analogous to the sigma 70-type promoter previously reported . Complementation of the P . aeruginosa sigX-disrupted mutant with plasmid-encoded OprF did not resolve the phenotypes associated with this mutant, which included a markedly reduced logarithmic-phase growth rate in rich medium (compared to that in minimal medium), further reduction of the growth rate in a low-osmolarity environment, secretion of an unidentified pigment, and increased sensitivity to the antibiotic imipenem . This indicates that SigX is involved in the regulation of other genes in P . aeruginosa . Disruption of the sigX gene in P . fluorescens also had an effect on the logarithmic-phase growth rate in rich medium . A conserved sigX gene was also identified in a Pseudomonas syringae isolate and six P . aeruginosa clinical isolates . Collectively, these data indicate that an ECF sigma factor plays a role in the regulation and expression of OprF and also affects other genes. J Appl Microbiol, 1999 Jul, 87(1), 80 - 90 Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54; Nielsen TH et al.; Pseudomonas fluorescens DR54 showed antagonistic properties against plant pathogenic Pythium ultimum and Rhizoctonia solani both in vitro and in planta . Antifungal activity was extractable from spent growth media, and fractionation by semi-preparative HPLC resulted in isolation of an active compound, which was identified as a new bacterial cyclic lipodepsipeptide, viscosinamide, using 1D and 2D 1H-, 13C-NMR and mass spectrometry . The new antibiotic has biosurfactant properties but differs from the known biosurfactant, viscosin, by containing glutamine rather than glutamate at the amino acid position 2 (AA2) . No viscosin production was observed, however, when Ps . fluorescens DR54 was cultured in media enriched with glutamate . In vitro tests showed that purified viscosinamide also reduced fungal growth and aerial mycelium development of both P . ultimum and R . solani . Viscosinamide production by Ps . fluorescens DR54 was tightly coupled to cell proliferation in the batch cultures, as the viscosinamide produced per cell mass unit approached a constant value . In batch cultures with variable initial C, N or P nutrient levels, there were no indications of elevated viscosinamide production during starvation or maintenance of the cultures in stationary phase . Analysis of cellular fractions and spent growth media showed that a major fraction of the viscosinamide produced remained bound to the cell membrane of Ps . fluorescens DR54 . The isolation, determination of structure and production characteristics of the new compound with both biosurfactant and antibiotic properties have promising perspectives for the application of Ps . fluorescens DR54 in biological control. Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1091 - 101 Pseudomonas libanensis sp . nov., a new species isolated from Lebanese spring waters; Dabboussi F et al.; The taxonomic position of eight fluorescent Pseudomonas isolates, from two Lebanese spring waters, which were previously recognized by numerical analysis as members of a new subcluster (subcluster Vb) was examined . Except for one strain, the new subcluster exhibited internal DNA hybridization values of 76-100%, and 9-53% hybridization was measured with the type or reference strains of other Pseudomonas species . The highest DNA binding value was found with Pseudomonas marginalis strains (37-53%) . The G+C content of the DNA of the type strain was 58 mol% . A comparison of 1322 nt of the 16S rRNA gene sequence of the strain representing subcluster Vb (CFML 96-195T) with the sequence of other strains of the genus Pseudomonas revealed that strain CFML 96-195T was part of the 'Pseudomonas fluorescens intrageneric cluster' . On the basis of the results of phenotypic, DNA-DNA and phylogenetic analyses, a new Pseudomonas species, Pseudomonas libanensis sp . nov., is proposed for the seven strains of subcluster Vb . The type strain is P . libanensis CFML 96-195T and has been deposited in the Collection de l'Institut Pasteur (Paris, France) as CIP 105460T . The P . libanensis strains are phenotypically and genotypically homogeneous and can be differentiated from most other fluorescent species by several phenotypic features . Differentiation of P . libanensis and Pseudomonas aeruginosa is based mainly on pyocyanin production; P . libanensis can be differentiated from P . fluorescens (all biovars) by alpha-aminobutyrate assimilation . The clinical significance of P . libanensis is unknown. Res Microbiol, 1999 Jun, 150(5), 303 - 16 Taxonomic study of bacteria isolated from Lebanese spring waters: proposal for Pseudomonas cedrella sp . nov . and P . orientalis sp . nov; Dabboussi F et al.; Deoxyribonucleic acid relatedness studies (S1 nuclease method) have shown that 15 strains isolated from three Lebanese spring waters, belonging to the genus Pseudomonas, formed two homogeneous DNA groups, with a within-group DNA relatedness ranging from 70 to 100% . These groups are referred to as Pseudomonas cedrella sp . nov . and Pseudomonas orientalis sp.nov . These strains were previously grouped on the basis of a numerical analysis in phenons Ve, Vd, Vg, and VI . DNA relatedness with 65 strains representing 24 species of the genus Pseudomonas sensu stricto was below 50% . The highest DNA binding value (50%) was found with P . marginalis species . A comparison of the complete 16S rRNA gene sequences of the strains representing the two new deoxyribonucleic acid hybridization groups, i.e., strains CFML 96-198T and CFML 96-170T, and the sequence of other strains of the genus Pseudomonas revealed that these strains (CFML 96-198T and CFML 96-170T) fell within the 'Pseudomonas fluorescens intrageneric cluster' . The G+C contents of the DNA of P . cedrella CIP 105541T and P . orientalis CIP 105540T were 59 and 60 mol%, respectively . The two species can be differentiated from each other by the fact that P . cedrella strains hydrolyze erythritol and D-lyxose . P . cedrella grouped together a total of nine strains from phenotypic groups Ve, Vg, and VI . P . orientalis grouped together six strains from both phenotypic groups Vd and Ve. J Food Prot, 1999 Apr, 62(4), 368 - 79 Different responses of planktonic and attached Bacillus subtilis and Pseudomonas fluorescens to sanitizer treatment; Lindsay D et al.; Three commercial sanitizers containing iodophor (I), peracetic acid/ hydrogen peroxide (PAH), or chlorhexidine gluconate (CG) were evaluated in vitro against planktonic and sessile Bacillus subtilis or Pseudomonas fluorescens cells grown in Standard One Nutrient Broth . Sessile cells were attached to stainless steel or polyurethane test surfaces . Planktonic and attached cells of both bacteria were enumerated by plate counts after sanitizer treatment for 1, 3, or 5 min . Sessile cells were dislodged from test surfaces by shaking them with beads . Cell morphologies were monitored by scanning electron microscopy (SEM) . Attached B . subtilis and P . fluorescens cells on both surface types were less susceptible to all three sanitizers than their planktonic counterparts . PAH, I, and CG were equally effective against planktonic P . fluorescens cells, which were reduced by 99.999% after 1, 3, and 5 min exposure . PAH was the only sanitizer effective against attached P . fluorescens cells on both surface types; it reduced counts by < or = 99.9% after 1, 3, and 5 min exposure . PAH was also the most effective sanitizer against planktonic B . subtilis cells, reducing counts by 99.9% after 1, 3, and 5 min . Sessile B . subtilis cells on both surface types were the least susceptible to all sanitizers; counts were reduced by only 99.5% or less after exposure to PAH for 5 min . SEM revealed that planktonic and attached cells of both bacteria exhibited symptoms of surface roughness, indentations, and shape distortions after treatment with any of the sanitizers. Arch Microbiol, 1999 May-Jun, 171(6), 424 - 9 Generation and initial characterization of Pseudomonas stutzeri KC mutants with impaired ability to degrade carbon tetrachloride; Sepulveda-Torres LC et al.; Under iron-limiting conditions, Pseudomonas stutzeri KC secretes a small but as yet unidentified factor that transforms carbon tetrachloride (CT) to CO2 and nonvolatile products when activated by reduction at cell membranes . Pseudomonas fluorescens and other cell types activate the factor . Triparental mating was used to generate kanamycin-resistant lux::Tn5 recombinants of strain KC . Recombinants were streaked onto the surface of agar medium plugs in microtiter plates and were then screened for carbon tetrachloride degradation by exposing the plates to gaseous 14C-carbon tetrachloride . CT+ recombinants generated nonvolatile 14C-labeled products, but four CT- recombinants did not generate significant nonvolatile 14C-labeled products and had lost the ability to degrade carbon tetrachloride . When colonies of P . fluorescens were grown next to colonies of CT+ recombinants and were exposed to gaseous 14C-carbon tetrachloride, 14C-labeled products accumulated around the P . fluorescens colonies, indicating that the factor secreted by CT+ colonies had diffused through the agar and become activated . When P . fluorescens was grown next to CT- colonies, little carbon tetrachloride transformation was observed, indicating a lack of active factor . Expression of lux reporter genes in three of the CT- mutants was regulated by added iron and was induced under the same iron-limiting conditions that induce carbon tetrachloride transformation in the wild-type. Appl Environ Microbiol, 1999 Jun, 65(6), 2294 - 9 Two-component transcriptional regulation of N-acyl-homoserine lactone production in Pseudomonas aureofaciens; Chancey ST et al.; Production of phenazine antibiotics by the biological control bacterium Pseudomonas aureofaciens 30-84 is regulated in part by the PhzI/PhzR N-acyl-homoserine lactone (AHL) response system (L . S . Pierson III, V . D . Keppenne, and D . W . Wood, J . Bacteriol . 176:3966-3974, 1994; D . W . Wood and L . S . Pierson III, Gene 168:49-53, 1996) . Two mutants, 30-84W and 30-84.A2, were isolated and were found to be deficient in the production of phenazine, protease, hydrogen cyanide (HCN), and the AHL signal N-hexanoyl-homoserine lactone . These mutants were not complemented by phzI, phzR, or the phenazine biosynthetic genes (phzFABCD) (L . S . Pierson III, T . Gaffney, S . Lam, and F . Gong, FEMS Microbiol . Lett . 134:299-307, 1995) . A 2.2-kb region of the 30-84 chromosome which fully restored production of all of these compounds in strain 30-84W was identified . Nucleotide sequence analysis of this region revealed a single open reading frame encoding a predicted 213-amino-acid protein which is very similar to the global response regulator GacA . Strain 30-84.A2 was not complemented by gacA or any cosmid from a genomic library of strain 30-84 but was complemented by gacS (formerly lemA) homologs from Pseudomonas fluorescens Pf-5 (N . Corbel and J . E . Loper, J . Bacteriol . 177:6230-6236, 1995) and Pseudomonas syringae pv . syringae B728a (E . M . Hrabek and D . K . Willis, J . Bacteriol . 174:3011-3020, 1992) . Transcription of phzR was not altered in either mutant; however, phzI transcription was eliminated in strains 30-84W and 30-84.A2 . These results indicated that the GacS/GacA two-component signal transduction system of P . aureofaciens 30-84 controls the production of AHL required for phenazine production by mediating the transcription of phzI . Addition of exogenous AHL did not complement either mutant for phenazine production, indicating that the GacS/GacA global regulatory system controls phenazine production at multiple levels . Our results reveal for the first time a mechanism by which a two-component regulatory system and an AHL-mediated regulatory system interact. J Appl Microbiol, 1999 May, 86(5), 817 - 26 Tolerance and biodegradation of m-toluate by Scots pine, a mycorrhizal fungus and fluorescent pseudomonads individually and under associative conditions; Sarand I et al.; The tolerance to, and degradation of m-toluate by Scots pine (Pinus sylvestris), a symbiotic mycorrhizal fungus (Suillus bovinus) and Pseudomonas fluorescens strains, with or without m-toluate-degrading capacity, was determined individually and in all symbiotic/associative plant-microbe combinations . Fungal survival on medium with m-toluate was increased in co-culture with the degradative bacterial strains on agar plates (up to 0.02%, w/v) . When fungi were grown in mycorrhizal association with Scots pine seedlings in test-tube microcosms containing expanded clay pellets and growth media, the fungus was able to withstand m-toluate concentrations up to 2.0%, w/v in all treatments . The seedling tolerance remained unaltered regardless of the presence or absence of mycorrhizal fungi or biodegradative bacteria . Reduction in m-toluate levels was only detected in treatments inoculated with bacterial strains harbouring TOL catabolic plasmids . The plant and fungus, alone or in mycorrhizal symbiosis, were unable to cleave m-toluate . The presence of easily available plant-derived carbon sources did not impede m-toluate degradation by the bacteria in the mycorrhizosphere. Appl Environ Microbiol, 1999 Jun, 65(6), 2794 - 7 A new biocatalyst for production of optically pure aryl epoxides by styrene monooxygenase from Pseudomonas fluorescens ST; Di Gennaro P et al.; We developed a biocatalyst by cloning the styrene monooxygenase genes (styA and styB) from Pseudomonas fluorescens ST responsible for the oxidation of styrene to its corresponding epoxide . Recombinant Escherichia coli was able to oxidize different aryl vinyl and aryl ethenyl compounds to their corresponding optically pure epoxides . The results of bioconversions indicate the broad substrate preference of styrene monooxygenase and its potential for the production of several fine chemicals. Appl Environ Microbiol, 1999 Jun, 65(6), 2429 - 38 Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas fluorescens biocontrol strains; Duffy BK et al.; Understanding the environmental factors that regulate the biosynthesis of antimicrobial compounds by disease-suppressive strains of Pseudomonas fluorescens is an essential step toward improving the level and reliability of their biocontrol activity . We used liquid culture assays to identify several minerals and carbon sources which had a differential influence on the production of the antibiotics 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT), and pyrrolnitrin and the siderophores salicylic acid and pyochelin by the model strain CHA0, which was isolated from a natural disease-suppressive soil in Switzerland . Production of PHL was stimulated by Zn2+, NH4Mo2+, and glucose; the precursor compound mono-acetylphloroglucinol was stimulated by the same factors as PHL . Production of PLT was stimulated by Zn2+, Co2+, and glycerol but was repressed by glucose . Pyrrolnitrin production was increased by fructose, mannitol, and a mixture of Zn2+ and NH4Mo2+ . Pyochelin production was increased by Co2+, fructose, mannitol, and glucose . Interestingly, production of its precursor salicylic acid was increased by different factors, i.e., NH4Mo2+, glycerol, and glucose . The mixture of Zn2+ and NH4Mo2+ with fructose, mannitol, or glycerol further enhanced the production of PHL and PLT compared with either the minerals or the carbon sources used alone, but it did not improve siderophore production . Extending fermentation time from 2 to 5 days increased the accumulation of PLT, pyrrolnitrin, and pyochelin but not of PHL . When findings with CHA0 were extended to an ecologically and genetically diverse collection of 41 P . fluorescens biocontrol strains, the effect of certain factors was strain dependent, while others had a general effect . Stimulation of PHL by Zn2+ and glucose was strain dependent, whereas PLT production by all strains that can produce this compound was stimulated by Zn2+ and transiently repressed by glucose . Inorganic phosphate reduced PHL production by CHA0 and seven other strains tested but to various degrees . Production of PLT but not pyrrolnitrin by CHA0 was also reduced by 100 mM phosphate . The use of 1/10-strength nutrient broth-yeast extract, compared with standard nutrient broth-yeast extract, amended with glucose and/or glycerol resulted in dramatically increased accumulations of PHL (but not PLT), pyochelin, and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow . The results of this study (i) provide insight into the biosynthetic regulation of antimicrobial compounds, (ii) limit the number of factors for intensive study in situ, and (iii) indicate factors that can be manipulated to improve bacterial inoculants. Microb Ecol, 1999 May, 37(4), 248 - 256 Effects of Pseudomonas fluorescens F113 on Ecological Functions in the Pea Rhizosphere Are Dependent on pH; Naseby DC et al.; > Abstract The aim of this microcosm study was to determine influence of the antibiotic 2,4-diacetylphloroglucinol (DAPG) on the effect of wild-type and functionally modified Pseudomonas fluorescens F113 strains in a sandy loam soil of pH 5.4 planted with pea (Pisum sativum var Montana) . The functional modification of strain F113 was a repressed production of DAPG, useful in plant disease control, creating the DAPG negative strain F113 G22; both were marked with a lacZY gene cassette . Lowering the soil pH to 4.4 significantly reduced the plant shoot and root weights and the root length, whereas the bacterial inocula had no significant effect . Both inocula significantly reduced the shoot/root ratio at pH 5.4, but this effect was not evident at the lowered or elevated (6.4) pH levels . The decrease in pH significantly increased the fungal and yeast colony-forming units from the rhizosphere (root extract), but did not affect the total bacterial c.f.u.'s . Inoculatioin with strain F113 in the pH 4.4 soil resulted in a significantly greater total bacterial population . The fungal and yeast c.f.u.'s were not significantly affected by the inocula at any pH studied . Increasing the pH significantly increased the indigenous Pseudomonas population in comparison to the reduced pH treatment and significantly increased both the introduced and total Pseudomonas populations . The antibiotic producing strain significantly reduced the total bacterial population and the NAGase activity (related to fungal activity) at pH 6.4 where the inocula population was the greatest . Alkaline phosphatase, phosphodiesterase, aryl sulfatase, beta-glucosidase, alkaline beta-galactosidase, and NAGase activities significantly increased with increasing in pH . The F113 inocula reduced the acid phosphatase activity at pH 5.4 and increased the acid beta-galactosidase activity over all the pH treatments . The results presented illustrate the variation in impact with soil pH, with implications for variability in efficacy of Pseudomonas fluorescens biocontrol agents with soil pH.http://link.springer-ny.com/link/service/journals/00248/bibs/37n4p248.html J Food Prot, 1999 May, 62(5), 543 - 6 Purification and characterization of three extracellular proteinases produced by Pseudomonas fluorescens INIA 745, an isolate from ewe's milk; Fernandez J et al.; Three proteinases were isolated from culture medium of Pseudomonas fluorescens INIA 745 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography . Optimal temperature for enzymatic activity was 45 degrees C for all three proteinases . The pH optimum of proteinases I and II was found to be 7.0, while that of proteinase III was 8.0 . Divalent metal ions like Cu2+, Co2+, Zn2+, Fe2+, and Hg2+ were inhibitory to proteinase activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect . The three enzymes were strongly inhibited by EDTA and 1,10-phenantroline and partially by cysteine . The three enzymes are metalloproteinases since they were inhibited by chelators and reactivated by Co2+, Mn2+, Cu2+, and Zn2+ . The Km values of proteinases I, II, and III for casein were calculated to be 3.2, 2.6, and 5.2 mg/ml, respectively . Proteinases II and III rapidly degraded beta-casein, with preference to alphas1-casein, whereas proteinase I hydrolyzed both casein fractions at a slow rate. FEMS Microbiol Lett, 1999 May 15, 174(2), 273 - 8 Lux-biosensor assessment of pH effects on microbial sorption and toxicity of chlorophenols; Sinclair GM et al.; Lux-marked bacterial biosensors and a commercial toxicity testing bacterial strain (Microtox) were exposed to 2,4-dichlorophenol (DCP) and the light output response measured . Increasing DCP concentrations caused a decrease in light output in all three biosensors with an order of sensitivity (in terms of luminescence decrease over the DCP concentration range) of Pseudomonas fluorescens < Escherichia coli < Microtox . Adsorption of DCP to E . coli was measured using uniformly ring labelled {14C}DCP and found to be very rapid . The effect of pH on toxicity and adsorption was also investigated . Low pH values increased the amount of DCP adsorbed to the cell and increased the toxicity of DCP. Biochemistry, 1999 May 11, 38(19), 6111 - 8 Electron transfer from quinohemoprotein alcohol dehydrogenase to blue copper protein azurin in the alcohol oxidase respiratory chain of Pseudomonas putida HK5; Matsushita K et al.; A blue copper protein was purified together with a type II quinohemoprotein alcohol dehydrogenase (ADH IIB) from the soluble fraction of Pseudomonas putida HK5 grown on n-butanol . The purified blue copper protein was shown to be azurin, on the basis of several properties such as its absorption maximum (623 nm), its low molecular mass (17 500 Da), its acidic nature (pI of 4.1), its relatively high redox potential (306 mV), the presence of an intramolecular disulfide bond, and N-terminal amino acid sequence homology with respect to azurins from other sources, especially from P . putida NCIB 9869 and Pseudomonas fluorescens . Direct electron transfer from ADH IIB to azurin was shown to occur at a rate of 48-70 s-1 . The apparent Km value of ADH IIB for azurin, determined by steady-state kinetics, was decreased several-fold by increasing the ionic strength . Furthermore, the extent of fluorescence quenching of ADH IIB due to the interaction with azurin was increased by increasing the ionic strength, but the binding constant for binding between ADH IIB and azurin was unchanged . The redox potential of azurin was increased 12 mV by incubation with ADH but not vice versa . Furthermore, the redox potential gap between ADH and azurin was increased from 102 to 126 mV by increasing the ionic strength . It is conceivable that a hydrophobic interaction is involved in the electron transfer between both proteins, and it is also suggested that the electron transfer may occur by a freely reversible on and off binding process but may not be related to the global binding process of both proteins . Thus, the results presented here strongly suggest that azurin works as an electron-transfer mediator in a PQQ-dependent alcohol oxidase respiratory chain in P . putida HK5. Appl Environ Microbiol, 1999 May, 65(5), 2032 - 4 A new sensitive bioassay for determination of microbially available phosphorus in water Lehtola MJ, Miettinen IT, Vartiainen T, Martikainen PJ. The content of assimilable organic carbon has been proposed to control the growth of microbes in drinking water . However, recent results have shown that there are regions where it is predominantly phosphorus which determines the extent of microbial growth in drinking waters . Even a very low concentration of phosphorus (below 1 &mgr;g of P liter-1) can promote extensive microbial growth . We present here a new sensitive method to determine microbially available phosphorus concentrations in water down to 0.08 &mgr;g of P liter-1 . The method is a bioassay in which the analysis of phosphorus in a water sample is based on maximum growth of Pseudomonas fluorescens P17 when the energy supply and inorganic nutrients, with the exception of phosphorus, do not limit bacterial growth . Maximum growth (CFU) in the water sample is related to the concentration of phosphorus with the factor 373,200 +/- 9,400 CFU/&mgr;g of PO4-P . A linear relationship was found between cell growth and phosphorus concentration between 0.05 to 10 &mgr;g of PO4-P liter-1 . The content of microbially available phosphorus in Finnish drinking waters varied from 0.1 to 10.2 &mgr;g of P liter-1 (median, 0.60 &mgr;g of P liter-1). Appl Microbiol Biotechnol, 1999 Mar, 51(3), 397 - 400 Containment of a genetically engineered microorganism during a field bioremediation application; Ford CZ et al.; A field release of a genetically engineered microorganism was performed at the Field Lysimeter Site on the Oak Ridge Reservation . Six large lysimeters were filled with soil that had been contaminated with a mixture of naphthalene, phenanthrene, and anthracene . A genetically engineered bacterial strain, Pseudomonas fluorescens HK44, was sprayed onto the surface of the soil during soil loading . This strain contains a fusion between the lux genes of Vibrio fischeri and the promoter for the lower pathway of naphthalene degradation, enabling the strain to become bioluminescent when it is degrading naphthalene . Release of the bacteria outside the lysimeters was monitored, using selective agar plates and one-stage Anderson air samplers . Although approximately 10(14) bacteria were sprayed during the loading process, escape was only detected sporadically; the highest incidence of bacterial escape was found when the relative humidity and wind speed were low. Arch Biochem Biophys, 1999 May 1, 365(1), 10 - 6 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL)--An enzyme of phenylpropanoid chain cleavage from Pseudomonas; Mitra A et al.; The enzyme 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), which catalyzes a hydration and two-carbon cleavage step in the degradation of 4-hydroxycinnamic acids, has been purified and characterized from Pseudomonas fluorescens strain AN103 . The enzyme is a homodimer and is active with three closely related substrates, 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA (Km values: 5.2, 1.6, and 2.4 microM, respectively), but not with cinnamoyl-CoA or with sinapinoyl-CoA . The abundance of the enzyme reflects a low catalytic center activity (2.3 molecules s-1 at 30 degrees C; 4-coumaroyl-CoA as substrate) . Biosci Biotechnol Biochem, 1999 Feb, 63(2), 293 - 7 Promotion of antibiotic production by high ethanol, high NaCl concentration, or heat shock in Pseudomonas fluorescens S272; Nakata K et al.; A stress imposed by a continuous feed of high ethanol, high NaCl concentration, or a high temperature shock increased antibiotic production by several times in Pseudomonas fluorescens S272 . A tentative bioassay showed that the stress caused about 40-fold elevation in the autoinducer activity . Addition of synthetic autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone or N-(3-oxohexanoyl)-L-homoserine lactone at a concentration of more than 100 micrograms/l to a non-stressed culture also increased the antibiotic production by several times . These results suggested that the antibiotic production in P . fluorescens S272 was regulated by N-acyl-homoserine lactone and the promotive effect by stress occurred through any function that increased the autoinducer production. Cryobiology, 1999 Mar, 38(2), 131 - 9 Identification of a novel ice-nucleating bacterium of Antarctic origin and its ice nucleation properties; Obata H et al.; A novel ice-nucleating bacterium (INB) was isolated from Ross Island, Antarctica . INBs could be isolated more frequently than was generally thought . INB strain IN-74 was found in the white colony group . Strain IN-74 was identified from its taxonomic characteristics as a novel INB, Pseudomonas antarctica IN-74 . When strain IN-74 was cultured aerobically in a medium consisting of the ice-nucleating broth (pH 7.0) for 6 days at 4 degrees C, the ice-nucleating activity of strain IN-74 cells was obtained . Strain IN-74 cells produced ice nuclei only at extremely low growth temperatures . The nuclei appeared to be less thermolabile than those of INB Pseudomonas fluorescens KUIN-1 . The freezing difference spectra in D2O and H2O at ice-nucleating temperature for strain IN-74 cells and conventional INBs (Pseudomonas fluorescens KUIN-1, Pseudomonas viridiflava KUIN-2, and Pseudomonas syringae C-9) exhibited different curves . Biotechnol Bioeng, 1998 Jun 5, 58(5), 554 - 9 Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones; Bornscheuer UT et al.; The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated . Mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones . Screening was performed by plating esterase producing colonies derived from mutation cycles onto minimal media agar plates containing indicator substances (neutral red and crystal violet) . Esterase-catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease caused by the released acid . Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester . One variant containing two mutations (A209D and L181V) stereoselectively hydrolyzed the ethyl ester resulting in 25% ee for the remaining ester . Biotechnol Bioeng, 1998 Jun 5, 58(5), 486 - 93 A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports Bastida A, Sabuquillo P, Armisen P, Fernandez-Lafuente R, Huguet J, Guisan JM. A number of bacterial lipases can be immobilized in a rapid and strong fashion on octyl-agarose gels (e.g., lipases from Candida antarctica, Pseudomonas fluorescens, Rhizomucor miehei, Humicola lanuginosa, Mucor javanicus, and Rhizopus niveus) . Adsorption rates in absence of ammonium sulfate are higher than in its presence, opposite to the observation for typical hydrophobic adsorption of proteins . At 10 mM phosphate, adsorption of lipases is fairly selective allowing enzyme purification associated with their reversible immobilization . Interestingly, these immobilized lipase molecules show a dramatic hyperactivation . For example, lipases from R . niveus, M . miehei, and H . lanuginosa were 6-, 7-, and 20-fold more active than the corresponding soluble enzymes when catalyzing the hydrolysis of a fully soluble substrate (0.4 mM p-nitrophenyl propionate) . Even higher hyperactivations and interesting changes in stereospecificity were also observed for the hydrolysis of larger soluble chiral esters (e.g . (R,S)-2-hydroxy-4-phenylbutanoic ethyl ester) . These results suggest that lipases recognize these "well-defined" hydrophobic supports as solid interfaces and they become adsorbed through the external areas of the large hydrophobic active centers of their "open and hyperactivated structure" . This selective interfacial adsorption of lipases becomes a very promising immobilization method with general application for most lipases . Through this method, we are able to combine, via a single and easily performed adsorption step, the purification, the strong immobilization, and a dramatic hyperactivation of lipases acting in the absence of additional interfaces, (e.g., in aqueous medium with soluble substrate) . Z Naturforsch {C}, 1999 Jan-Feb, 54(1-2), 105 - 9 Identification and cloning of a gene locus encoding peptid