Ps. fluorescens

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 peptide synthetase of Pseudomonas fluorescens by two sets of PCR primers; Rajendran N; A chromosomal locus encoding biosynthetic genes for a putative peptide synthetase of Pseudomonas fluorescens was identified and cloned . To achieve this, two sets of degenerated oligonucleotide primers KAGGA:SGTTG and TGD:LGG were used in PCR . These primers were selected based on highly conserved units of known peptide synthetases involved in adenylation and thiolation regions of Bacillus subtilis . The discrete amplified bands from PCR ca . 300 bp for KAGGA:SGTTG and ca . 500 bp for TGD:LGG proved to be integral part of the genomic DNA of P . fluorescens were cloned and sequenced . Sequence alignments of both fragments confirmed the putative peptide synthetase genes in P . fluorescens . The present study describes the identification and cloning of peptide synthetase genes of P . fluorescens, which can be used to identify a genetic locus encoding peptide synthetase in other microbial species.

J Bacteriol, 1999 Apr, 181(7), 2166 - 74
Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas fluorescens Pf-5; Nowak-Thompson B et al.; Ten genes (plt) required for the biosynthesis of pyoluteorin, an antifungal compound composed of a bichlorinated pyrrole linked to a resorcinol moiety, were identified within a 24-kb genomic region of Pseudomonas fluorescens Pf-5 . The deduced amino acid sequences of eight plt genes were similar to the amino acid sequences of genes with known biosynthetic functions, including type I polyketide synthases (pltB, pltC), an acyl coenzyme A (acyl-CoA) dehydrogenase (pltE), an acyl-CoA synthetase (pltF), a thioesterase (pltG), and three halogenases (pltA, pltD, and pltM) . Insertions of the transposon Tn5 or Tn3-nice or a kanamycin resistance gene in each of these genes abolished pyoluteorin production by Pf-5 . The presumed functions of the eight plt products are consistent with biochemical transformations involved in pyoluteorin biosynthesis from proline and acetate precursors . Isotope labeling studies demonstrated that proline is the primary precursor to the dichloropyrrole moiety of pyoluteorin . The deduced amino acid sequence of the product of another plt gene, pltR, is similar to those of members of the LysR family of transcriptional activators . pltR and pltM are transcribed divergently from the pltLABCDEFG gene cluster, and a sequence with the characteristics of a LysR binding site was identified within the 486-bp intergenic region separating pltRM from pltLABCDEFG . Transcription of the pyoluteorin biosynthesis genes pltB, pltE, and pltF, assessed with transcriptional fusions to an ice nucleation reporter gene, was significantly greater in Pf-5 than in a pltR mutant of Pf-5 . Therefore, PltR is proposed to be a transcriptional activator of linked pyoluteorin biosynthesis genes.

Eur J Biochem, 1999 Feb, 260(1), 120 - 6
Purification, characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5; Nakano H et al.; 6-Hydroxynicotinate 3-monooxygenase, a membrane-bound, 42-kDa monomeric enzyme from Pseudomonas fluorescens TN5 was purified and characterized . The enzyme catalyzes the oxidative decarboxylation of 6-hydroxynicotinate and depends on O2, NADH and FAD with the holoenzyme containing 1 M of FAD per 1 M of enzyme . The isolated enzyme was used for the synthesis of 2,5-dihydroxypyridine, a precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy . A 1.8-kbp DNA fragment, which contains the ORF encoding 6-hydroxynicotinic acid 3-monooxygenase, was cloned, sequenced and expressed in Escherichia coli . The deduced 385 amino acid sequence of the cloned ORF is in agreement with the enzyme molecular mass, amino acid sequence of an internal peptide, contains a putative FAD-binding site and is homologous to similar flavoproteins such as salicylate 1-monoxygenase.

Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 544 - 6
Crystallization and preliminary X-ray analysis of arabinanase A from Pseudomonas fluorescens subspecies cellulosa; Scott M et al.; Crystals of 1,5-alpha-arabinanase A from Pseudomonas fluorescens subspecies cellulosa have been obtained by vapour diffusion . The crystals belong to the space group P6122 with unit-cell parameters a = b = 91.6, c = 179.4 A with one molecule in the asymmetric unit . The native crystals and, to a much greater extent, heavy-atom soaked crystals are sensitive to radiation which necessitates cryocooling . Suitable cryocooling conditions have been established, though a shrinkage of the unit cell is observed, with a = b = 88.8 and c = 176.9 A.

J Microencapsul, 1999 Mar-Apr, 16(2), 215 - 29
Rhizobacteria microencapsulation: properties of microparticles obtained by spray-drying; Amiet-Charpentier C et al.; Rhizobacteria Pseudomonas fluorescens-putida were microencapsulated in Eudragit by spray-drying . These microparticles are subsequently included in seed coating or pelleting material . The survival of the bacterial cell in microparticles were studied under different levels of relative humidity (RH): 0, 33, 55 and 100% . The protective effects of silica, present in certain formulations, were demonstrated at the relative humidities of 33 and 55% . The release of the encapsulated bacteria was also studied over time . The release was fast, the bacteria being observed at 15 min immersion of the Eudragit microparticles in an aqueous-buffer medium at 20 degrees C . This result, related to the physicochemical character of the coating polymer, showed that water was the triggering element for the release of rhizobacteria . Compatibility studies between two film-forming agents used for seed coatings and the encapsulated bacteria, as well as wettability measures of tableted microparticles, were carried out . The bacterial survival was good with the seed coating agent, Sepiret 1039G, and the wettability measurements of agglomerated microparticles were in accord with the rapid release of the microencapsulated bacteria . The application of microparticles containing rhizobacteria on seeds can now be considered for preliminary trials.

Ann N Y Acad Sci, 1998 Dec 13, 864, 81 - 6
Novel double bond-transferring hydroxylation reaction involved in microbial metabolism of eugenol; Furukawa H et al.; We isolated a eugenol-degrading bacterium, Pseudomonas fluorescens E118 . This strain produced a novel enzyme, eugenol dehydrogenase, which catalyzes the conversion of eugenol into coniferyl alcohol . The enzyme was purified from the eugenol-induced cells of P . fluorescens E118 . The purified enzyme appeared to be homogeneous, judging from the analysis of polyacrylamide gel electrophoresis . The enzyme was a 68-kDa protein composed of two different subunits (alpha subunit, 10 kDa; and beta subunit, 58 kDa) . The enzyme exhibited a cytochrome c-like absorption spectrum . The alpha subunit corresponded to cytochrome c . The enzyme catalyzed the dehydrogenation of 4-alkylphenol into the corresponding alkyl 1-(4-hydroxyphenyl)-alcohol derivatives . The reaction products were isolated and identified physicochemically . The enzyme catalyzed the enantioselective hydroxylation of p-alkylphenols . p-Ethylphenol and p-propylphenol were converted to S-(-)-p-(1-hydroxyphenyl)ethanol and S-(-)-p(1-hydroxyphenyl)propanol, respectively.

Appl Environ Microbiol, 1999 Mar, 65(3), 969 - 73
Inhibition of vibrio anguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatment of fish; Gram L et al.; To study the possible use of probiotics in fish farming, we evaluated the in vitro and in vivo antagonism of antibacterial strain Pseudomonas fluorescens strain AH2 against the fish-pathogenic bacterium Vibrio anguillarum . As iron is important in virulence and bacterial interactions, the effect of P . fluorescens AH2 was studied under iron-rich and iron-limited conditions . Sterile-filtered culture supernatants from iron-limited P . fluorescens AH2 inhibited the growth of V . anguillarum, whereas sterile-filtered supernatants from iron-replete cultures of P . fluorescens AH2 did not . P . fluorescens AH2 inhibited the growth of V . anguillarum during coculture, independently of the iron concentration, when the initial count of the antagonist was 100 to 1, 000 times greater that of the fish pathogen . These in vitro results were successfully repeated in vivo . A probiotic effect in vivo was tested by exposing rainbow trout (Oncorynchus mykiss Walbaum) to P . fluorescens AH2 at a density of 10(5) CFU/ml for 5 days before a challenge with V . anguillarum at 10(4) to 10(5) CFU/ml for 1 h . Some fish were also exposed to P . fluorescens AH2 at 10(7) CFU/ml during the 1-h infection . The combined probiotic treatment resulted in a 46% reduction of calculated accumulated mortality; accumulated mortality was 25% after 7 days at 12 degrees C in the probiotic-treated fish, whereas mortality was 47% in fish not treated with the probiont.

Can J Microbiol, 1998 Nov, 44(11), 1086 - 93
Naphthalene uptake by a Pseudomonas fluorescens isolate; Whitman BE et al.; The uptake of naphthalene has been investigated in the metabolizing cells of Pseudomonas fluorescens utilizing {1-14C}naphthalene . The uptake displayed an affinity constant (Kt) of 11 microM and a maximal velocity (Vmax) of 17 nmol.h-1.mg-1 cellular dry weight . Naphthalene uptake was not observed in a mutant strain, TG-5, which was unable to utilize naphthalene as a sole source of carbon for growth . Uptake was significantly inhibited (approximately 90%) by the presence of growth-inhibiting levels of either azide or 2,4-dinitrophenol and was sensitive to the presence of structural analogues of naphthalene . The intracellular levels of ATP were not significantly reduced by the presence of either azide or 2,4-dinitrophenol . The presence of alpha-naphthol was found to noncompetitively inhibit naphthalene uptake, displaying a Ki of 0.041 microM . It is concluded that the first step in the utilization of naphthalene by Pseudomonas fluorescens is its transport into the cell by a specific energy-linked transport system.

FEBS Lett, 1999 Jan 29, 443(3), 251 - 5
Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase . Implications for NADPH recognition and structural stability; Eppink MH et al.; Phe161 and Arg166 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding {1} . To study their role in more detail, Phe161 and Arg166 were selectively changed by site-directed mutagenesis . F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH . The catalytic properties of R166K are similar to those of the native enzyme . R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD . The crystal structure of substrate complexed F161A at 2.2 A is indistinguishable from the native enzyme, except for small changes at the site of mutation . The crystal structure of substrate complexed R166S at 2.0 A revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain . It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH.

J Biotechnol, 1999 Jan 22, 67(2-3), 99 - 112
Biodegradation of benzene, toluene, ethylbenzene, and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor; Shim H et al.; A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P . fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams . The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied . In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics . However, for immobilized cells, the degradation rate was much higher than that of free cells . Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (> 1000 mg l-1) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene . Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions . Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor . Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l-1 . Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.

Biosci Biotechnol Biochem, 1998 Nov, 62(11), 2233 - 5
Molecular cloning and analysis of a lipase gene from Pseudomonas fluorescens No . 33; Kumura H et al.; The gene encoding an extracellular lipase from Pseudomonas fluorescens No . 33 was cloned and sequenced . A single open reading frame consisting of 1,428 nucleotides that encoded a mature protein of 476 amino acids was recognized . Sequence analysis showed that the deduced molecular weight of 50,209 agreed with the molecular weight of the purified lipase as measured by SDS-PAGE and the lipase lacked a signal peptide . The presence of a repeating motif, GXXGXDXXX, suggested that the lipase might be exported and secreted via a system that involves the ATP-binding cassette protein.

Biosci Biotechnol Biochem, 1998 Nov, 62(11), 2091 - 7
Purification and characterization of a novel cold-regulated protein from an ice-nucleating bacterium, Pseudomonas fluorescens KUIN-1; Obata H et al.; The psychrotrophic ice-nucleating bacterium, Pseudomonas fluorescens KUIN-1 respond to a decrease in temperature with the induction of proteins that are classified as cold shock proteins (CSPs) . We found the function of a 26-kDa protein of the CSPs in the strain KUIN-1 . In strain KUIN-1, a cold shock from 18 to 4 degrees C induced the synthesis of the 26-kDa protein . By analysis with SDS-PAGE, it was then demonstrated that the 26-kDa protein was produced by the cells after treatment at 4 degrees C . The 26-kDa protein was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies (QA52, phenyl Superose, Superose 12, and Mono Q) . The purified 26-kDa protein is composed of 6 subunits of 26.5-kDa with a molecular mass of approximately 159-kDa according to gel filtration and SDS-PAGE . The N-terminal sequence of the 26-kDa protein was Gln-Ala-Ala-Tyr-Tyr-Pro-Ala-His-His-His-Gln- Gln-Val-Gln-Gln-His-Trp-Gly-His-His- . Specifically, 26-kDa protein of the CSPs of strain KUIN-1 was very effective in protecting the cold-labile enzyme, lactate dehydrogenase against denaturation by freezing . The characteristics of 26-kDa protein are analogous to the cold-regulated protein of the plants.

J Hosp Infect, 1999 Jan, 41(1), 23 - 8
Contaminated lithium heparin bottles as a source of pseudobacteraemia due to Pseudomonas fluorescens; Namnyak S et al.; Pseudobacteraemia might be responsible for up to 50% of all positive blood cultures and its early recognition is important in order to avoid unnecessary treatment with antibiotics and delay in the search for the true cause of the fever . We describe pseudobacteraemia outbreak of Pseudomonas fluorescens related to contaminated lithium heparin bottles in a paediatric ward . Twelve patients were involved in this outbreak from December 1996-January 1997 . All patients had no clinical evidence of sepsis, nevertheless most children were treated with antibiotics . Blood collection bottles were suspected as source of pseudobacteraemia and only lithium heparin bottles were found to be contaminated with P . fluorescences indistinguishable from the blood isolates taken from these children . Withdrawal of these bottles led to the termination of the pseudobacteraemia . Following discussion with the manufacturer, the contaminated batch of lithium heparin bottles was sent back for testing, and replaced with bottles containing dried lithium heparin . A hazard report was sent to the Medical Devices Agency (MDA) . In order to minimize the possibility of this problem occurring again, the manufacturer has informed MDA that all lithium heparin solution is to be filtered to 0.2 micron prior to issue, in order to minimize bacterial contamination . Continued monitoring after the pseudobacteraemia showed no isolates of P . fluorescens from the blood of paediatric patients.

Appl Environ Microbiol, 1999 Feb, 65(2), 813 - 21
Simultaneous monitoring of cell number and metabolic activity of specific bacterial populations with a dual gfp-luxAB marker system; Unge A et al.; A dual marker system was developed for simultaneous quantification of bacterial cell numbers and their activity with the luxAB and gfp genes, encoding bacterial luciferase and green fluorescent protein (GFP), respectively . The bioluminescence phenotype of the luxAB biomarker is dependent on cellular energy status . Since cellular metabolism requires energy, bioluminescence output is directly related to the metabolic activity of the cells . By contrast, GFP fluorescence has no energy requirement . Therefore, by combining these two biomarkers, total cell number and metabolic activity of a specific marked cell population could be monitored simultaneously . Two different bacterial strains, Escherichia coli DH5alpha and Pseudomonas fluorescens SBW25, were chromosomally tagged with the dual marker cassette, and the cells were monitored under different conditions by flow cytometry, plate counting, and luminometry . During log-phase growth, the luciferase activity was proportional to the number of GFP-fluorescent cells and culturable cells . Upon entrance into stationary phase or during starvation, luciferase activity decreased due to a decrease in cellular metabolic activity of the population, but the number of GFP-fluorescing cells and culturable cells remained relatively stable . In addition, we optimized a procedure for extraction of bacterial cells from soil, allowing GFP-tagged bacteria in soil samples to be quantitated by flow cytometry . After 30 days of incubation of P . fluorescens SBW25::gfp/lux in soil, the cells were still maintained at high population densities, as determined by GFP fluorescence, but there was a slow decline in luciferase activity, implicating nutrient limitation . In conclusion, the dual marker system allowed simultaneous monitoring of the metabolic activity and cell number of a specific bacterial population and is a promising tool for monitoring of specific bacteria in situ in environmental samples.

Appl Environ Microbiol, 1999 Feb, 65(2), 477 - 82
Screening, nucleotide sequence, and biochemical characterization of an esterase from Pseudomonas fluorescens with high activity towards lactones; Khalameyzer V et al.; A genomic library of Pseudomonas fluorescens DSM 50106 in a lambdaRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using alpha-naphthyl acetate and Fast Blue RR . A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced . Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family . The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase . However, esterase activity was not induced by growing of P . fluorescens DSM 50106 in the presence of several cyclic ketones . The esterase gene was fused to a His tag and expressed in E . coli . The gene product was purified by zinc ion affinity chromatography and characterized . Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated . The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43 degreesC . The showed highest purified enzyme activities towards lactones . The activity increased from gamma-butyrolactone (18.1 U/mg) to epsilon-caprolactone (21.8 U/mg) to delta-valerolactone (36.5 U/mg) . The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.

Nat Toxins, 1998, 6(2), 61 - 5
Elevation of alanine amino transferase and aspartate amino transferase produced by pyoverdin, a photolabile pigment of Pseudomonas fluorescens; Eraso AJ et al.; The effect of three forms pyoverdin on mouse liver was studied . Significant increases of alanine amino transferase (ALT) and aspartate amino transferase (AST) were obtained in mice after ingestion of water with forms A and C . The effect on liver was more evident with A than with C . Pyoverdin was purified by means of salt saturation, solvent extractions and ion-exchange chromatography . Fluorescent peaks obtained in the presence of light were different from those eluted under dark conditions . The relative amounts of pyoverdin A, B and C varied when dark purification procedure was employed . Form A decreased while C increased in the absence of light . Optimum conditions for C were in the dark without iron . When C was exposed to light, it changed to form A . Fast Atom Bombardment (FAB) mass spectrometry of pyoverdin form C gave a form at M+ = 1324 m.u., which is 9 m.u . less than pyoverdin purified in the presence of light . The results suggest that light can influence pyoverdin stability and toxicity.

Appl Biochem Biotechnol, 1998 Aug, 74(2), 85 - 93
Fermentation of milk permeate by proteolytic bacteria for protease production; Ali AA et al.; Kinetics of cell growth and protease production by four proteolytic bacterial strains, namely, Bacillus subtilis EMCC 1020, Bacillus megaterium EMCC 1057, Serratia marcescens EMCC 1247, and Pseudomonas fluorescens EMCC 1221, in milk permeate, compared with other fermentation media, were studied . The pH values, lactose utilization, and biochemical oxygen demand (BOD) reduction in milk permeate also were investigated . The four strains were able to grow in milk permeate, and to produce considerable amounts of protease, reaching 289 (EMCC 1020), 252 (EMCC 1057), 263 (EMCC 1247), and 212 (EMCC 1221) U/mL after 30 h of fermentation . The growth and enzyme activity of the four strains were greater in milk permeate than those in other fermentation media . The protease-producing bacteria were able to utilize lactose in milk permeate with values ranging from 37.62 to 54.97% and to reduce the BOD of milk permeate by 50.59-63.65% . Milk permeate proved to be the best medium for enzyme production by all organisms examined.

J Bacteriol, 1998 Dec, 180(24), 6635 - 41
The two-component regulators GacS and GacA influence accumulation of the stationary-phase sigma factor sigmaS and the stress response in Pseudomonas fluorescens Pf-5; Whistler CA et al.; Three global regulators are known to control antibiotic production by Pseudomonas fluorescens . A two-component regulatory system comprised of the sensor kinase GacS (previously called ApdA or LemA) and GacA, a member of the FixJ family of response regulators, is required for antibiotic production . A mutation in rpoS, which encodes the stationary-phase sigma factor sigmaS, differentially affects antibiotic production and reduces the capacity of stationary-phase cells of P . fluorescens to survive exposure to oxidative stress . The gacA gene of P . fluorescens Pf-5 was isolated, and the influence of gacS and gacA on rpoS transcription, sigmaS levels, and oxidative stress response of Pf-5 was determined . We selected a gacA mutant of Pf-5 that contained a single nucleotide substitution within a predicted alpha-helical region, which is highly conserved among the FixJ family of response regulators . At the entrance to stationary phase, sigmaS content in gacS and gacA mutants of Pf-5 was less than 20% of the wild-type level . Transcription of rpoS, assessed with an rpoS-lacZ transcriptional fusion, was positively influenced by GacS and GacA, an effect that was most evident at the transition between exponential growth and stationary phase . Mutations in gacS and gacA compromised the capacity of stationary-phase cells of Pf-5 to survive exposure to oxidative stress . The results of this study provide evidence for the predominant roles of GacS and GacA in the regulatory cascade controlling stress response and antifungal metabolite production in P . fluorescens.

J Bacteriol, 1998 Dec, 180(24), 6551 - 6
Sequence diversity of the oprI gene, coding for major outer membrane lipoprotein I, among rRNA group I pseudomonads; De Vos D et al.; The sequence of oprI, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on Pseudomonas aeruginosa and Pseudomonas fluorescens . Within the P . aeruginosa species, oprI sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96 . The oprI sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with P . aeruginosa oprI) . An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found . A clustering according to the respective presence and/or positions of the HaeIII, PvuII, and SphI sites could also be obtained . A sequence cluster analysis showed a rather widespread distribution of P . fluorescens isolates . All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the oprI gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads.

Microbiology, 1998 Nov, 144 ( Pt 11), 3119 - 26
Siderotyping of fluorescent pseudomonads: characterization of pyoverdines of Pseudomonas fluorescens and Pseudomonas putida strains from Antarctica; Meyer JM et al.; Five independent fluorescent pseudomonad isolates originating from Antarctica were analysed for their pyoverdine systems . A pyoverdine-related siderotyping, which involved pyoverdine-induced growth stimulation, pyoverdine-mediated iron uptake, pyoverdine analysis by electrophoresis and isoelectric focusing, revealed three different pyoverdine-related siderotypes among the five isolates . One siderotype, including Pseudomonas fluorescens 1W and P . fluorescens 10CW, was identical to that of P . fluorescens ATCC 13525 . Two other strains, P . fluorescens 9AW and Pseudomonas putida 9BW, showed identical pyoverdine-related behaviour to each other, whereas the fifth strain, P . fluorescens 51W, had unique features compared to the other strains or to a set of 12 fluorescent Pseudomonas strains used as comparison material . Elucidation of the structure of the pyoverdines produced by the Antarctic strains supported the accuracy of the siderotyping methodology by confirming that pyoverdines from strains 1W and 10CW had the same structures as the P . fluorescens ATCC 13525 pyoverdine, whereas the 9AW and 9BW pyoverdines are probably identical with the pyoverdine of P . fluorescens strain 244 . Pyoverdine from strain 51W appeared to be a novel pyoverdine since its structure was different from all previously established pyoverdine structures . Together with the conclusion that the Antarctic Pseudomonas strains have no special features at the level of their pyoverdines and pyoverdine-mediated iron metabolism compared to worldwide strains, the present work demonstrates that siderotyping provides a rapid means of screening for novel pyoverdines.

J Bacteriol, 1998 Dec, 180(23), 6352 - 63
Combined physical and genetic map of the Pseudomonas putida KT2440 chromosome; Ramos-Diaz MA et al.; A combined physical and genetic map of the Pseudomonas putida KT2440 genome was constructed from data obtained by pulsed-field gel electrophoresis techniques (PFGE) and Southern hybridization . Circular genome size was estimated at 6.0 Mb by adding the sizes of 19 SwaI, 9 PmeI, 6 PacI, and 6 I-CeuI fragments . A complete physical map was achieved by combining the results of (i) analysis of PFGE of the DNA fragments resulting from digestion of the whole genome with PmeI, SwaI, I-CeuI, and PacI as well as double digestion with combinations of these enzymes and (ii) Southern hybridization analysis of the whole wild-type genome digested with different enzymes and hybridized against a series of probes obtained as cloned genes from different pseudomonads of rRNA group I and Escherichia coli, as P . putida DNA obtained by PCR amplification based on sequences deposited at the GenBank database, and by labeling of macrorestriction fragments of the P . putida genome eluted from agarose gels . As an alternative, 10 random mini-Tn5-Km mutants of P . putida KT2440 were used as a source of DNA, and the band carrying the mini-Tn5 in each mutant was identified after PFGE of a series of complete chromosomal digestions and hybridization with the kanamycin resistance gene of the mini-Tn5 as a probe . We established a circular genome map with an average resolution of 160 kb . Among the 63 genes located on the genetic map were key markers such as oriC, 6 rrn loci (rnnA to -F), recA, ftsZ, rpoS, rpoD, rpoN, and gyrB; auxotrophic markers; and catabolic genes for the metabolism of aromatic compounds . The genetic map of P . putida KT2440 was compared to those of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens SBW25 . The chromosomal backbone revealed some similarity in gene clustering among the three pseudomonads but differences in physical organization, probably as a result of intraspecific rearrangements.

Infect Immun, 1998 Dec, 66(12), 5854 - 61
Modulation of expression of the ToxR regulon in Vibrio cholerae by a member of the two-component family of response regulators; Wong SM et al.; The ToxRS system in Vibrio cholerae plays a central role in the modulation of virulence gene expression in response to environmental stimuli . An integration of multiple signalling inputs mediated by ToxR, -S, and -T controls virulence gene expression leading to cholera toxin (CT) production . Recently, we identified a new virulence locus, varA (virulence associated regulator), in classical V . cholerae O1 that positively controls transcription of tcpA, the major subunit of the toxin-coregulated pilus (TCP) and the production of CT, two key factors in cholera pathogenesis . The varA locus is a homolog of gacA (originally described for the soil organism Pseudomonas fluorescens), which encodes a conserved global regulator belonging to the family of two-component signal transducing molecules . GacA homologs in a number of diverse gram-negative pathogenic bacterial species have been implicated in controlling the production of diverse virulence factors . varA mutants showed reduced levels of tcpA message and TcpA protein, lacked visible signs of autoagglutination (a phenotype associated with functional TCP), produced decreased levels of CT, and were attenuated in colonizing infant mice . Transcription of varA appears to be independent of ToxR, and overexpression of the regulators tcpPH and toxT from plasmids in the varA mutant restored wild-type levels of CT production and the ability to autoagglutinate . varA represents an additional modulating factor in the coordinate expression of virulence factors in V . cholerae.

Mol Microbiol, 1998 Nov, 30(3), 547 - 55
Different residues in periplasmic domains of the CcmC inner membrane protein of Pseudomonas fluorescens ATCC 17400 are critical for cytochrome c biogenesis and pyoverdine-mediated iron uptake; Gaballa A et al.; The inner membrane protein CcmC (CytA) of Pseudomonas fluorescens ATCC17400, which has homologues in several bacteria and plant mitochondria, is needed for the biogenesis of cytochrome c . A CcmC-deficient mutant is also compromised in the production and utilization of pyoverdine, the high-affinity fluorescent siderophore . A topological model for CcmC, based on the analysis of alkaline phosphatase fusions, predicts six membrane-spanning regions with three periplasmic loops . Site-directed mutagenesis was used in order to assess the importance of some periplasm-exposed residues, conserved in all CcmC homologues, for cytochrome c biogenesis, and pyoverdine production/utilization . Despite the conservation of the residues His-61, Val-62 and Pro-63 in the first periplasmic loop, and Leu-184, His-185 and Gln-186 in the third periplasmic loop, their simultaneous replacement with Ala only partially affected cytochrome c biogenesis and pyoverdine production/utilization . Simultaneous replacements of residues Trp-115 and Gly-116 in the second periplasmic loop substantially affected pyoverdine production/utilization but not cytochrome c production . An Ala substitution of Asp-127, in the second periplasmic loop, resulted in decreased production of cytochrome c, slower growth in conditions of anaerobiosis and reduced pyoverdine production . On the other hand, a mutation in Trp-126, also in the second periplasmic loop, totally suppressed the production of cytochrome c, whereas it had no effect on the production and utilization of pyoverdine . These results show a differential involvement of amino acid residues in periplasmic domains of CcmC in cytochrome c biogenesis and pyoverdine production/utilization.

J Colloid Interface Sci, 1998 Dec 1, 208(1), 23 - 33
Effects of Substratum Topography on Bacterial Adhesion; Scheuerman TR et al.; The effect of substratum topography on bacterial surface colonization was studied using a chemically homogeneous silicon coupon . "Grooves" 10 microm deep and 10, 20, 30, and 40 microm wide were etched on the coupon perpendicular to the direction of flow . Flow (Re = 5.5) of a bacterial suspension (10(8) cells/ml) was directed through a parallel plate flow chamber inverted on a confocal microscope . Images were collected in real time to obtain rate and endpoint colonization data for each of three strains of bacteria: Pseudomonas aeruginosa and motile and nonmotile Pseudomonas fluorescens . A higher velocity experiment (Re = 16.6) and an abiotic control using hydrophilic, negatively charged microspheres were also performed . Using a colloidal deposition expression, the initial rates of attachment were compared . P . aeruginosa attached at a higher rate than P . fluorescens mot+ which attached at a higher rate than P . fluorescens mot- . For all bacteria the rate was independent of groove size and was greatest on the downstream edges of the grooves . Only the motile organisms were found in the bottoms of the grooves . A higher fluid velocity resulted in an increase in the initial rate of attachment . In contrast, there was no adhesion of the beads . Attachment of the bacteria appears to be predominated by transport from the bulk phase to the substratum .

Microb Pathog, 1998 Oct, 25(4), 197 - 201
Are bacterial proteins part of the matrix of kidney stones?
Daskalova S, Kostadinova S, Gauster D, Prohaska R, Ivanov A.
An extracellular protein, produced from Pseudomonas fluorescens strain D with molecular mass of 41.5 kDa was partially purified . Its first 12 amino acid sequence shows strong similarity to a sequence reported to belong to a protein isolated from a urate-calcium oxalate stone (Binnette & Binnette, Scan Microsc1994; 2: 233-239) . A possible involvement of bacterial proteins in stone matrix is discussed .

J Food Prot, 1998 Oct, 61(10), 1341 - 6
Effect of Pseudomonas fluorescens on beef discoloration and oxymyoglobin oxidation in vitro; Chan WK et al.; The relationship between bacterial growth and oxymyoglobin oxidation in vitro and in meat was studied . In the in vitro study, oxymyoglobin was combined with Pseudomonas fluorescens or sterile nutrient broth (control) in an airtight vessel . P . fluorescens samples showed greater metmyoglobin formation and oxygen consumption than controls . The P . fluorescens population in the reaction vessels was correlated with metmyoglobin formation (r = 0.85, P < 0.05) and oxygen consumption (r = 0.91, P < 0.05) . When P . fluorescens and oxymyoglobin were combined in an airtight vessel, reducing the headspace from 13 ml and 9 ml to 3 ml resulted in greater metmyoglobin formation (P < 0.05) . In the meat study, beef cores prepared from longissimus lumborum were inoculated with P . fluorescens (10(7) CFU/cm2) or sterile peptone water (control), packaged under 1% O2 (+99% N2), air, or 100% O2 and stored at 4 degrees C . Inoculated beef cores showed higher bacterial loads and metmyoglobin formation than their respective controls during 10 h storage in 1% O2, 3 days in air, and 7 days in 100% O2 (P < 0.05) . This finding indicated that P . fluorescens could accelerate beef discoloration . Overall, studies demonstrated that oxygen consumption concomitant with P . fluorescens growth decreased partial oxygen pressure, which accelerated oxymyoglobin oxidation.

J Food Prot, 1998 Oct, 61(10), 1336 - 40
Bacterial tracking in a dairy production system using phenotypic and ribotyping methods; Ralyea RD et al.; A systematic sampling plan was designed to collect raw and pasteurized milk samples throughout a single-raw milk source, dairy-processing operation experiencing reduced product shelf lives due to bacterial contamination . The objectives were to track bacterial contamination sources throughout a complete dairy production system and use this information to reduce bacterial spoilage losses in processed fluid products . Over a 5-week period, 233 bacterial isolates were collected, representative of different colony morphologies on psychrotrophic bacteria count (PBC) plates . Forty-five isolates (19%) were obtained from pasteurized milk and 188 (81%) were isolated from raw product . Thirty isolates were identified as Pseudomonas spp . by Gram stain and biochemical methods . Of these, 27 (90%) were postpasteurization isolates and 3 (10%) were raw milk isolates . Automated ribotyping revealed that raw and pasteurized Pseudomonas fluorescens isolates were indistinguishable (similarity index > 0.93), suggesting the possibility of postpasteurization contamination with bacteria from raw product . In the plant, filler nozzles were identified as the primary reservoirs of postpasteurization contamination . Nozzle replacement produced significantly lower finished-product PBCs at 7 days postprocessing (> 4-log reduction) and extended fluid product shelf life.

Appl Environ Microbiol, 1998 Nov, 64(11), 4452 - 9
Accumulation of alpha-keto acids as essential components in cyanide assimilation by Pseudomonas fluorescens NCIMB 11764; Kunz DA et al.; Pyruvate (Pyr) and alpha-ketoglutarate (alphaKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L . Chen and D . A . Kunz, FEMS Microbiol . Lett . 156:61-67, 1997) . The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 micromol of cyanide removed min-1 ml of culture fluid-1) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM . The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by 13C nuclear magnetic resonance spectroscopy . Both the Pyr and alpha-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates . Radiotracer experiments showed that CO2 (and NH3) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct . Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate . Pyr and alphaKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate . The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role.

Mikrobiologiia, 1998 Jul-Aug, 67(4), 505 - 11
{Characteristics of Pseudomonas fluorescens IMV 1433 (Biovar I) lipopolysaccharide}; Veremeichenko SN; The lipopolysaccharide of strain Pseudomonas fluorescens IMV 1433 (biovar I) was isolated and investigated . 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, hexadecenoic, and octadecenoic acids were identified in the composition of lipid A . The total amount of hydroxy acids makes up 60% . In the hydrolysate of lipid A, components of the were revealed hydrophilic skeleton--glucosamine and phosphoethanolamine--were revealed . Glucose, galactose, rhamnose, glucosamine, galactosamine, alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulosonic acid were identified in the core of the lipopolysaccharide molecule . O-specific polysaccharide chains of the lipopolysaccharide are built from repeating trisaccharide units consisting of the residues of 2-acetamido-2,6-dideoxy-L-glucose, 2-acetamido-2,6-dideoxy-D-glucose and 4-acetamido-4,6-dideoxy-D-galactose . The lipopolysaccharide studied is similar to the lipopolysaccharide of P . fluorescens IMV 1152 (biovar I) in the structure of lipid A and core oligosaccharide, and identical to it in the structure of the O-chain (this identity correlates with the high level of the serological relationship of the strains).

Biochem Mol Biol Int, 1998 Sep, 46(1), 43 - 54
Chemical inactivation of bacterial GABA aminotransferase; Tunnicliff G et al.; The effects of three potential irreversible inhibitors of gamma-aminobutyrate aminotransferase from Pseudomonas fluorescens were studied in order to throw more light on the nature of the active site of the enzyme . The thiol group reagent mercuric chloride inactivated the enzyme in a concentration-dependent manner . Inhibition kinetics were consistent with a simple bimolecular reaction . The second-order rate constant was 4.2 x 10(3) +/- 0.61 M-1 sec-1 . In contrast to either of the substrates, the cofactor pyridoxal 5'-phosphate could protect the enzyme from the inhibition, suggesting cysteinyl residues are important for cofactor binding at the active site . p-Chloromercuribenzoic acid produced a similar inactivation of the enzyme . 4-Amino-2-fluorobutanoic acid also inhibited enzymic activity but in this case the inhibition was reversible and competitive with respect to gamma-aminobutyric acid (GABA) . The inhibitor constant (Ki) was 0.83 +/- 0.44 mM . We found no evidence that this fluorinated analogue of GABA could act as a substrate for the enzyme.

Indian J Exp Biol, 1998 Jul, 36(7), 693 - 8
Maintenance of a Pseudomonas fluorescens plasmid in heterologous hosts: metabolic burden as a more reliable variable to predict plasmid instability; Chandrasekaran S et al.; The stability of a large, multiresistance plasmid, pSCL of P . fluorescens CAS102 was studied in Pseudomonas putida and E . coli under various non-stress conditions . Both the strains lost the plasmid within 25 days when repeatedly subcultured in LB broth without any antibiotic . The transformants survived in sterile soil and water without any marked reduction in the viability . In sterile soil, P . putida lost 93% and E . coli, 98% of their plasmid containing population in 30 days, while in sterile water the plasmid loss was 92.5% and 97% respectively . The two variables, viz . the efficiency of plasmid-partitioning during cell division and measurement of relative specific growth rates of plasmid-plus and plasmid-minus cells which are used to predict plasmid instability cannot be used to predict plasmid loss during starvation . The utility of a third variable, viz . the metabolic burden due to plasmid maintenance in predicting plasmid instability in different hosts is discussed . The rate of plasmid loss was found to be comparatively faster in E . coli than in P . putida . The biosynthetic burden due to plasmid maintenance was also more in E . coli than in P . putida when compared to the plasmid-plus and plasmid-minus cells of the two strains which was evident from the increased nutrient uptake rates (glucose, O2, and amino acid) and increased protein content of the plasmid-plus cells of E . coli . From the results, a correlation could be found between the degree of metabolic burden and the rate of plasmid loss . The reliability of metabolic burden, to predict plasmid instability versus the relative specific growth rates is discussed.

FEMS Microbiol Lett, 1998 Sep 15, 166(2), 171 - 6
Reverse transcription-PCR analysis of the regulation of ethylbenzene dioxygenase gene expression in Pseudomonas fluorescens CA-4; Corkery DM et al.; Pseudomonas fluorescens strain CA-4 is a bioreactor isolate previously characterised by the presence of a side chain oxidation pathway for ethylbenzene breakdown . In this report a second pathway involving ethylbenzene ring dioxygenation has been identified in this strain . We examine here second substrate inhibition of the genes encoding the initial enzymes of this pathway, using reverse transcription (RT)-PCR . The genes of the ring-dioxygenation have been cloned and sequenced . They exhibit near identity to the gene clusters encoding the aromatic ring dioxygenase enzymes of two previously described isopropyl degrading strains, Pseudomonas sp . strain JR1 and P . fluorescens IP01 . This dioxygenase pathway appears to be the major pathway for ethylbenzene degradation in this strain . The expression of these genes appears to be affected by the presence of second carbon substrates . Using RT-PCR we demonstrate that the negative effect of glutamate present in the growth medium together with ethylbenzene on the rate of ethylbenzene metabolism is mediated at the transcriptional level on the ethylbenzene dioxygenase genes.

Res Microbiol, 1997 May, 148(4), 355 - 64
Production of substituted naphthalene dihydrodiols by engineered Escherichia coli containing the cloned naphthalene 1,2-dioxygenase gene from Pseudomonas fluorescens N3; Gennaro PD et al.; Naphthalene dioxygenase, a key enzyme in the dihydroxylation of naphthalene, is encoded by the plasmid pN3, responsible for naphthalene metabolism in Pseudomonas fluorescens N3 . The naphthalene dioxygenase, including all the sequences for its expression and the regulatory region, has been localized on the 4.3-kb HindIII-ClaI fragment and on the 3.5-kb HindIII fragment of the plasmid pN3, by Southern analysis using as probes nahA and nahR genes, the homologous genes of the plasmid NAH7 from Pseudomonas putida G7 . We cloned in Escherichia coli JM109 the dioxygenase gene and its regulatory region and developed an efficient bacterial system inducible by salicylic acid, able to produce dihydrodiols . E . coli containing recombinant plasmids carrying the dioxygenase gene were analysed for their potential as a biocatalytic tool to produce dihydrodiols from different naphthalenes with the substituent on the aromatic ring at the alpha or beta position . The dihydrodiols, identified by HPLC (high-performance liquid chromatography) and 1H-NMR (nuclear magnetic resonance) were produced with yields ranging from 50 to 94% . The degree of bioconversion efficiency depends on the nature and the position of the substituent and indicates the broad substrate specificity of this dioxygenase and its potential for the production of a wide variety of fine chemicals.

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 129 - 31
Crystallization and preliminary X-ray diffraction studies of a family 26 endo-beta-1,4 mannanase (ManA) from Pseudomonas fluorescens subspecies cellulosa; Scott M et al.; Crystals of an endo-beta-1,4-mannanase (1,4-beta-D-mannohydrolase, E . C . 3.2.1.78) from Pseudomonas fluorescens sub species cellulosa have been grown by the hanging-drop technique at 291 K over a period of one to two weeks to maximal dimensions of 0.17 x 0.17 x 0.25 mm . These crystals belong to the space group R32 (or R3) with cell dimensions of a = b = 155.4 and c = 250.8 A (hexagonal setting) and contain three (six) molecules in the asymmetric unit . The crystals diffract to at least 3.2 A using a laboratory source and are suitable for structure determination.

Int J Food Microbiol, 1998 Aug 18, 43(1-2), 105 - 13
Concentration of carbon dioxide in the water-phase as a parameter to model the effect of a modified atmosphere on microorganisms; Devlieghere F et al.; The effect of modified atmosphere packaging can mainly be attributed to the bacteriostatic action of CO2 . The dissolved CO2 in the water-phase of a food product is strongly dependent on several intrinsic and extrinsic parameters and will determine the effectiveness of a modified atmosphere packaging configuration . The effect of pH, gas/product ratio, initial %CO2 in the gas-phase, lard content and storage temperature on the amount of dissolved CO2 was screened in a preliminary experiment . The initial CO2-concentration in the gas-phase and the gas/product ratio turned out to be the two major factors determining the amount of dissolved CO2 . The initial pH also determined significantly the final CO2-concentration in the broth . Temperature and lard content were shown to have only a minor effect on the amount of dissolved CO2 compared to the above mentioned parameters . This demonstrates the importance of the packaging configuration in the effectiveness of a modified atmosphere . In a second step, a model was constructed to predict the amount of dissolved carbon dioxide in modified BHI-broth as a function of the gas/product ratio, the initial CO2-concentration and the temperature by means of Response Surface Methodology (RSM) . A second equation was also derived based on Henry's law and was shown to be a powerful tool in the quantification of the effect of intrinsic and extrinsic parameters on the CO2-solubility in food products . The possibility of the use of the concentration of dissolved CO2 in the water-phase as a determinative factor for the inhibitory effect of modified atmospheres was examined on Pseudomonas fluorescens . Growth curves at 7 degrees C of P . fluorescens in different packaging configurations (initial %CO2 and gas/product ratio) resulting in equal amounts of dissolved CO2 were compared . P . fluorescens was shown to be similarly inhibited by equal amounts of dissolved CO2-concentrations, independent of the packaging configuration . This demonstrates the potential of the application of the concentration of dissolved CO2 in the water-phase as a parameter to characterise a modified atmosphere and its inhibition of certain microorganisms.

Appl Environ Microbiol, 1998 Oct, 64(10), 3563 - 9
Secondary metabolite- and endochitinase-dependent antagonism toward plant-pathogenic microfungi of pseudomonas fluorescens isolates from sugar beet rhizosphere
Nielsen MN, Sorensen J, Fels J, Pedersen HC.
Forty-seven isolates representing all biovars of Pseudomonas fluorescens (biovars I to VI) were collected from the rhizosphere of field-grown sugar beet plants to select candidate strains for biological control of preemergence damping-off disease . The isolates were tested for in vitro antagonism toward the plant-pathogenic microfungi Pythium ultimum and Rhizoctonia solani in three different plate test media . Mechanisms of fungal inhibition were elucidated by tracing secondary-metabolite production and cell wall-degrading enzyme activity in the same media . Most biovars expressed a specific mechanism of antagonism, as represented by a unique antibiotic or enzyme production in the media . A lipopeptide antibiotic, viscosinamide, was produced independently of medium composition by P . fluorescens bv . I, whereas the antibiotic 2, 4-diacetylphloroglucinol was observed only in glucose-rich medium and only in P . fluorescens bv . II/IV . Both pathogens were inhibited by the two antibiotics . Finally, in low-glucose medium, a cell wall-degrading endochitinase activity in P . fluorescens bv . I, III, and VI was the apparent mechanism of antagonism toward R . solani . The viscosinamide-producing DR54 isolate (bv . I) was shown to be an effective candidate for biological control, as tested in a pot experiment with sugar beet seedlings infested with Pythium ultimum . The assignment of different patterns of fungal antagonism to the biovars of P . fluorescens is discussed in relation to an improved selection protocol for candidate strains to be used in biological control.

Prog Nucleic Acid Res Mol Biol, 1998, 61, 211 - 41
Structure and function analysis of Pseudomonas plant cell wall hydrolases; Hazlewood GP et al.; Hydrolysis of the major structural polysaccharides of plant cell walls by the aerobic soil bacterium Pseudomonas fluorescens subsp . cellulosa is attributable to the production of multiple extracellular cellulase and hemicellulase enzymes, which are the products of distinct genes belonging to multigene families . Cloning and sequencing of individual genes, coupled with gene sectioning and functional analysis of the encoded proteins have provided a detailed picture of structure/function relationships and have established the cellulase-hemicellulase system of P . fluorescens subsp . cellulosa as a model for the plant cell wall degrading enzyme systems of aerobic cellulolytic bacteria . Cellulose- and xylan-degrading enzymes produced by the pseudomonad are typically modular in structure and contain catalytic and noncatalytic domains joined together by serine-rich linker sequences . The cellulases include a cellodextrinase; a beta-glucan glucohydrolase and multiple endoglucanases, containing catalytic domains belonging to glycosyl hydrolase families 5, 9, and 45; and cellulose-binding domains of families II and X, both of which are present in each enzyme . Endo-acting xylanases, with catalytic domains belonging to families 10 and 11, and accessory xylan-degrading enzymes produced by P . fluorescens subsp . cellulosa contain cellulose-binding domains of families II, X, and XI, which act by promoting close contact between the catalytic domain of the enzyme and its target substrate . A domain homologous with NodB from rhizobia, present in one xylanase, functions as a deacetylase . Mananase, arabinanase, and galactanase produced by the pseudomonad are single domain enzymes . Crystallographic studies, coupled with detailed kinetic analysis of mutant forms of the enzyme in which key residues have been altered by site-directed mutagenesis, have shown that xylanase A (family 10) has 8-fold alpha/beta barrel architecture, an extended substrate-binding cleft containing at least six xylose-binding pockets and a calcium-binding site that protects the enzyme from thermal inactivation, thermal unfolding, and attack by proteinases . Kinetic studies of mutant and wild-type forms of a mannanase and a galactanase from P . fluorescens subsp . cellulosa have enabled the catalytic mechanisms and key catalytic residues of these enzymes to be identified.

J Microencapsul, 1998 Sep-Oct, 15(5), 639 - 59
Microencapsulation of rhizobacteria by spray-drying: formulation and survival studies; Amiet-Charpentier C et al.; This research deals with the microencapsulation of the bacteria Pseudomonas fluorescens-putida, using the spray-drying technique . These bacteria act as a plant growth stimulator and the microparticles are subsequently included in the seed coating or pelleting material . The objective was to maintain living encapsulated bacteria for a minimum of 5-6 months . Three polymers were tested: a methacrylic copolymer from the Eudragit range, an ethylcellulose and a modified starch . A silica additive to the spraying food was also examined . The granulometric distribution and the morphology of the microparticles were studied and the residual moisture was measured after each bacteria survival control test . The best results were obtained with the methacrylic copolymer Eudragit, particularly for microparticles collected in the cone of the spray-drying chamber . The mean diameter of the microspheres was 44.6 microns, 85% of these particles having a size between 6.2 and 84.4 microns . The bacterial survival time of a particular strain incorporated in these microparticles, strain M3.1, was one year . A relationship was found between the bacterial survival and the microspheres' residual moisture, the best survival of the stored bacteria being observed when the residual moisture was around 25%.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 813 - 9
Phylogenetic relationships of Pseudomonas putida strains deduced from the nucleotide sequences of gyrB, rpoD and 16S rRNA genes; Yamamoto S et al.; Phylogenetic analysis of 20 Pseudomonas strains (Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas chlororaphis) was conducted by using the nucleotide sequences of the genes for 16S RNA, DNA gyrase B subunit (gyrB) and RNA polymerase delta 70 factor (rpoD), which have been determined by the direct sequencing of PCR-amplified fragments . On the basis of gyrB and rpoD sequences, these strains were split into two major clusters: one including the type strain of P . putida and all biovar A strains and the other including all P . putida biovar B strains, P . fluorescens stains and the P . chlororaphis strain . In the phylogenetic tree reconstructed from the 16S rRNA sequences included variable regions, P . Putida biovar A and B strains were not separated into two independent clusters, whereas in the phylogenetic tree reconstructed from the 16S rRNA sequences excluding the variable region sequences, these strains were separated into P . putida biovar A and biovar B clusters . The pairwise distances estimated from the variable regions of 16S rRNA correlated poorly with the synonymous distances estimated from the gyrB and rpoD genes . On the other hand, a highly significant correlation was observed between the pairwise distances estimated from the non-variable regions of 16S rRNA and the synonymous distances from gyrB and rpoD genes . Consequently, only the 16S rRNA sequences in the non-variable regions should be used for the phylogenetic analysis . The gyrB and rpoD analyses showed the necessity for the reclassification of P . putida biovar B strains.

Plant Cell, 1998 Sep, 10(9), 1571 - 80
A novel signaling pathway controlling induced systemic resistance in Arabidopsis; Pieterse CM et al.; Plants have the ability to acquire an enhanced level of resistance to pathogen attack after being exposed to specific biotic stimuli . In Arabidopsis, nonpathogenic, root-colonizing Pseudomonas fluorescens bacteria trigger an induced systemic resistance (ISR) response against infection by the bacterial leaf pathogen P . syringae pv tomato . In contrast to classic, pathogen-induced systemic acquired resistance (SAR), this rhizobacteria-mediated ISR response is independent of salicylic acid accumulation and pathogenesis-related gene activation . Using the jasmonate response mutant jar1, the ethylene response mutant etr1, and the SAR regulatory mutant npr1, we demonstrate that signal transduction leading to P . fluorescens WCS417r-mediated ISR requires responsiveness to jasmonate and ethylene and is dependent on NPR1 . Similar to P . fluorescens WCS417r, methyl jasmonate and the ethylene precursor 1-aminocyclopropane-1-carboxylate were effective in inducing resistance against P . s . tomato in salicylic acid-nonaccumulating NahG plants . Moreover, methyl jasmonate-induced protection was blocked in jar1, etr1, and npr1 plants, whereas 1-aminocyclopropane-1-carboxylate-induced protection was affected in etr1 and npr1 plants but not in jar1 plants . Hence, we postulate that rhizobacteria-mediated ISR follows a novel signaling pathway in which components from the jasmonate and ethylene response are engaged successively to trigger a defense reaction that, like SAR, is regulated by NPR1 . We provide evidence that the processes downstream of NPR1 in the ISR pathway are divergent from those in the SAR pathway, indicating that NPR1 differentially regulates defense responses, depending on the signals that are elicited during induction of resistance.

Mikrobiologiia, 1998 May-Jun, 67(3), 333 - 7
{Effect of water pollution by oil and oil products on barrier functions of bacterial cell cytoplasmic membranes}; Fomchenkov VM et al.; The effects of oil, diesel fuel, and kerosene on the electroorientational spectra and osmo-optical characteristics of bacterial cells were studied . Electroorientational spectra were found to be affected over the entire frequency range studied; changes in low-frequency (< 100 kHz) electroorientation were related to alterations in the cell surface, and those in high-frequency electroorientation, to the impairment of the barrier function of the plasma membrane . The membranotropic activity of petroleum products was also demonstrated by the osmo-optical method . Of nine bacterial species studied, Pseudomonas fluorescens VKM B-894, P . oleovorans VKM B-1522, and P . stutzeri VKM B-903 were most susceptible to the membranotropic action of kerosene; P . putida VKM B-1292 was the most resistant . Other bacterial strains studied were moderately sensitive.

Carbohydr Res, 1998 Jan, 306(1-2), 297 - 303
Somatic antigens of pseudomonads: structure of the O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247; Shashkov AS et al.; The O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247, was studied by acid hydrolysis, GLC-MS and 1H and 13C NMR spectroscopy, including 1D and 2D NOE, 2D hybrid TOCSY and ROESY (TORO), and 2D H-detected heteronuclear multiple-bond correlation (HMBC) experiments . The polysaccharide was found to contain L-rhamnose, 3.6-dideoxy-3-{(S)-3-hydroxybutyramido}-D-glucose (D-Qui3NHb), 2-acetamido- 2,4,6-trideoxy-4-{(S)-3-hydroxybutyramido-D-glucose (D-QuiNAc4NHb) and 2-acetamido-2- deoxy-D-galacturonic acid (D-GalNAcA) . Partial acid hydrolysis of the polysaccharide resulted in a non-reducing GalNAcA-->QuiNAc4NHb disaccharide with the 3-hydroxybutyryl group glycosylated intramolecularly by the QuiN4N residue . The following structure of the tetrasaccharide repeating unit of the polysaccharide was established:-->4) -alpha-D-GalpNAcA-(1-->3)- alpha-D-QuipNAc4NHb-(1-->2)-beta-D-Quip3NHb-(1-->2)-alpha-L- Rhap(1-->.

Appl Environ Microbiol, 1998 Aug, 64(8), 3123 - 6
Effect of humic fractions and clay on biodegradation of phenanthrene by a Pseudomonas fluorescens strain isolated from soil; Ortega-Calvo JJ et al.; The mineralization of phenanthrene in pure cultures of a Pseudomonas fluorescens strain, isolated from soil, was measured in the presence of soil humic fractions and montmorillonite . Humic acid and clay, either separately or in combination, shortened the acclimation phase . A higher mineralization rate was measured in treatments with humic acid at 100 microg/ml . Humic acid at 10 microg/ml stimulated the transformation only in the presence of 10 g of clay per liter . We suggest that sorption of phenanthrene to these soil components may result in a higher concentration of substrate in the vicinity of the bacterial cells and therefore may increase its bioavailability.

J Food Prot, 1998 Jul, 61(7), 844 - 8
Capacitance microbiology as a means of determining the quantity of spoilage bacteria on fish fillets; Russell SM; An experiment was conducted to determine if a method for enumeration of Pseudomonas fluorescens in less than 11 h could be used to predict potential spoilage of fresh fish of for species . In each of three separate replications (Rep), five boneless fillets from each species of fish, including rainbow trout (RT), Atlantic salmon (AS), red grouper (RG), and tilapia (T) were obtained fresh from a retail outlet . For each species, six 25-g samples of fish flesh were asceptically removed from each fillet, placed into a polyethylene bag, and stored at 3 degrees C for 0, 1, 2, 3, 4, or 5 days . After storage, samples were analyzed for psychotrophic plate count (PPC), Pseudomonas fluorescens plate counts (PFPC), and P . fluorescens capacitance detection times (PDT) and subjectively evaluated for odor (ODOR) . PPC gradually increased on all fish species as storage time increased . In most cases, PFPC decreased slightly and then progressively increased as storage time increased . In Reps 1 and 2, PDT decreased gradually (indicating an increase in bacteria); however, in Rep 3, PDT were erratic and difficult to interpret . Odor increased gradually throughout the storage period for all fish species . Linear correlations (R2 > 0.80) were observed between PPC and day of storage (DAY) for all fish species and Reps except for RT and RG in Rep 3 . PFPC correlated (R2 > 0.70) to DAY for all fish except RT in Rep 3 and RG in Rep 2 . PDT was negatively correlated to DAY for RT and T in Rep 1 and for all fish in Rep 2 . Odor scores were highly correlated (R2 > or = 0.84) to DAY for all fish tested . PPC and PDT were negatively correlated for RT in Reps 1 and 2, AS in Rep 2, and T in Reps 1 and 2 . Because results can be obtained in < 12 h, the capacitance procedure with further refinement may provide an excellent alternative to conducting PPC as a means of predicting potential spoilage of fish such that the fillets of inferior quality (i.e., those that will spoil rapidly) may be sent to distribution outlets that are known to move fish products quickly and are able to sell the fish before it spoils.

Mol Plant Microbe Interact, 1998 Aug, 11(8), 763 - 71
Role of the O-antigen of lipopolysaccharide, and possible roles of growth rate and of NADH:ubiquinone oxidoreductase (nuo) in competitive tomato root-tip colonization by Pseudomonas fluorescens WCS365; Dekkers LC et al.; Colonization-defective, transposon-induced mutants of the efficient root colonizer Pseudomonas fluorescens WCS365 were identified with a gnotobiotic system . Most mutants were impaired in known colonization traits, i.e., prototrophy for amino acids, motility, and synthesis of the O-antigen of LPS (lipopolysaccharide) . Mutants lacking the O-antigen of LPS were impaired in both colonization and competitive growth whereas one mutant (PCL1205) with a shorter O-antigen chain was defective only in colonization ability, suggesting a role for the intact O-antigen of LPS in colonization . Eight competitive colonization mutants that were not defective in the above-mentioned traits colonized the tomato root tip well when inoculated alone, but were defective in competitive root colonization of tomato, radish, and wheat, indicating they contained mutations affecting host range . One of these eight mutants (PCL1201) was further characterized and contains a mutation in a gene that shows homology to the Escherichia coli nuo4 gene, which encodes a subunit of one of two known NADH:ubiquinone oxidoreductases . Competition experiments in an oxygen-poor medium between mutant PCL1201 and its parental strain showed a decreased growth rate of mutant PCL1201 . The requirement of the nuo4 gene homolog for optimal growth under conditions of oxygen limitation suggests that the root-tip environment is micro-aerobic . A mutant characterized by a slow growth rate (PCL1216) was analyzed further and contained a mutation in a gene with similarity to the E . coli HtrB protein, a lauroyl transferase that functions in lipid A biosynthesis.

Nature, 1998 Jul 2, 394(6688), 69 - 72
Adaptive radiation in a heterogeneous environment; Rainey PB et al.; Successive adaptive radiations have played a pivotal role in the evolution of biological diversity . The effects of adaptive radiation are often seen, but the underlying causes are difficult to disentangle and remain unclear . Here we examine directly the role of ecological opportunity and competition in driving genetic diversification . We use the common aerobic bacterium Pseudomonas fluorescens, which evolves rapidly under novel environmental conditions to generate a large repertoire of mutants . When provided with ecological opportunity (afforded by spatial structure), identical populations diversify morphologically, but when ecological opportunity is restricted there is no such divergence . In spatially structured environments, the evolution of variant morphs follows a predictable sequence and we show that competition among the newly evolved niche-specialists maintains this variation . These results demonstrate that the elementary processes of mutation and selection alone are sufficient to promote rapid proliferation of new designs and support the theory that trade-offs in competitive ability drive adaptive radiation.

Gene, 1998 Jul 17, 215(1), 19 - 27
The mannitol utilization genes of Pseudomonas fluorescens are regulated by an activator: cloning, nucleotide sequence and expression of the mtlR gene; Brunker P et al.; A plasmid with the galK gene under control of the promoter of the mannitol utilization genes (mtl) from Pseudomonas fluorescens DSM 50106 was constructed to isolate the mtl regulatory gene . An Escherichia coli galK- mtl- strain with this plasmid was used to screen a genomic library of P . fluorescens for the presence of the regulatory gene by plating on McConkey agar plates supplemented with galactose and mannitol . Clones carrying the regulatory gene were isolated and by complemention assays, deletion analysis and DNA sequencing an open reading frame (mtlR) of 906nt identified encoding the regulator . The deduced protein MtlR with a calculated molecular mass of 34.7kDa showed a low overall similarity to several other regulatory proteins of the XylS/AraC family . When mtlR was cloned and expressed in E . coli, the protein was produced as inclusion bodies . Complete denaturation followed by subsequent slow refolding led to low amounts of active protein . The activity was shown in gel mobility shift assays by binding of MtlR to a DNA fragment containing the promoter/operator region of the P . fluorescens mtl genes.

FEBS Lett, 1998 Jun 16, 429(3), 312 - 6
All three surface tryptophans in Type IIa cellulose binding domains play a pivotal role in binding both soluble and insoluble ligands; Nagy T et al.; The three surface tryptophans of the Type IIa cellulose binding domain of Pseudomonas fluorescens subsp . cellulosa xylanase A (CBD(XYLA)) were independently mutated to alanine, to create the mutants W13A, W49A and W66A . The three mutant proteins were purified, and their capacity to bind to a variety of ligands was determined . The mutant proteins have native-like structures but exhibited much weaker affinity for crystalline and amorphous cellulose and for cellohexaose than the wild type . These data indicate that all three tryptophans are important for binding to cellulose, and support a model in which the three tryptophans form an aromatic strip on the surface of the protein that binds to a single cellulose.

Appl Environ Microbiol, 1998 Jul 1, 64(7), 2634 - 8
Effect of Insertion Site and Metabolic Load on the Environmental Fitness of a Genetically Modified Pseudomonas fluorescens Isolate; De Leij FAAM et al.; An isolate of Pseudomonas fluorescens (SBW25) was modified with different marker genes (lacZY, aph-1, and xylE) . These marker genes were inserted singly or in combination into two separate (1 Mbp apart) and presumably nonessential sites (-6- and Ee) on the chromosome of SBW25 . This allowed the production of a range of genetically modified SBW25 variants that differed with respect to insertion site of the marker genes and metabolic burden . The environmental fitness of the different SBW25 variants was tested in soil, in the rhizosphere of wheat and pea, and on the phylloplane of wheat . Reduced environmental fitness of the different variants was mainly attributed to the extra metabolic burden of novel gene expression, whereas choice of insertion site was of little significance . Changes in environmental fitness were dependent on the environmental conditions; an environment, such as soil, with a low microbial carrying capacity had a negative effect on the environmental fitness of variants with a large metabolic load . In environments with a larger carrying capacity, such as the rhizosphere of pea, environmental fitness of variants with a large metabolic load was not significantly different from that of variants with a smaller metabolic burden.

J Mol Biol, 1998 Jun 19, 279(4), 889 - 900
Structural investigation of the cofactor-free chloroperoxidases; Hofmann B et al.; The structures of cofactor-free haloperoxidases from Streptomyces aureofaciens, Streptomyces lividans, and Pseudomonas fluorescens have been determined at resolutions between 1.9 A and 1.5 A . The structures of two enzymes complexed with benzoate or propionate identify the binding site for the organic acids which are required for the haloperoxidase activity . Based on these complexes and on the structure of an inactive variant, a reaction mechanism is proposed for the halogenation reaction with peroxoacid and hypohalous acid as reaction intermediates . Comparison of the structures suggests that a specific halide binding site is absent in the enzymes but that hydrophobic organic compounds may fit into the active site pocket for halogenation at preferential sites .

Arch Microbiol, 1998 Jul, 170(1), 1 - 7
Functional citric acid cycle in an arcA mutant of Escherichia coli during growth with nitrate under anoxic conditions; Prohl C et al.; The operation of the citric acid cycle of Escherichia coli during nitrate respiration (anoxic conditions) was studied by measuring end products and enzyme activities . Excretion of products other than CO2, such as acetate or ethanol, was taken as an indication for a non-functional cycle . From glycerol, approximately 0.3 mol acetate was produced; the residual portion was completely oxidized, indicating the presence of a partially active citric acid cycle . In an arcA mutant devoid of the transcriptional regulator ArcA, glycerol was completely oxidized with nitrate as an electron acceptor, demonstrating derepression and function of the complete pathway . Glucose, on the other hand, was excreted mostly as acetate by the wild-type and by the arcA mutant . During growth on glucose, but not on glycerol, activities of succinate dehydrogenase and of 2-oxoglutarate dehydrogenase were missing nearly completely . Thus, the previously described strong repression of the citric acid cycle during nitrate respiration occurs only during growth on glucose and is the effect of anaerobic and, more important, of glucose repression . In Pseudomonas fluorescens (but not Pseudomonas stutzeri), a similar decrease of citric acid cycle function during anaerobic growth with nitrate was found, indicating a broad distribution of this regulatory principle.

Biochemistry, 1998 Jun 16, 37(24), 8783 - 9
The catalytic mechanism of kynureninase from Pseudomonas fluorescens: evidence for transient quinonoid and ketimine intermediates from rapid-scanning stopped-flow spectrophotometry; Phillips RS et al.; The reaction of Pseudomonas fluorescens kynureninase with L-kynurenine and L-alanine has been examined using rapid-scanning stopped-flow spectrophotometry . Mixing kynureninase with 0.5 mM L-kynurenine results in formation of a quinonoid intermediate, with lambdamax = 494 nm, within the dead time (ca . 2 ms) of the stopped-flow mixer . This intermediate then decays rapidly, with k = 743 s-1, and this rate constant is reduced to 347 s-1 in {2H}H2O, suggesting that protonation of this intermediate by a solvent exchangeable proton takes place . Rapid quench experiments demonstrate that covalent changes in the cofactor occur, as pyridoxal 5'-phosphate is converted to pyridoxamine 5'-phosphate in about 30 mol % within 5 ms after mixing . Under single turnover conditions in the reaction of kynureninase with l-kynurenine, a transient shoulder absorbing at 335 nm is observed that may be a pyruvate ketimine intermediate . In contrast, the reaction of kynureninase with 0.5 mM l-kynurenine in the presence of 10 mM benzaldehyde results in the formation of a quinonoid intermediate (k = 67.4 s-1) with a very strong absorbance peak at 496 nm . The reaction of L-alanine with kynureninase exhibits the rapid formation (386 s-1 at 0.1 M) of an external aldimine intermediate absorbing at 420 nm, followed by slower formation of a quinonoid intermediate with a peak at 500 nm (k = 2.5 s-1) . The 420 nm peak then decays slowly with concomitant formation of a peak at 320 nm corresponding to a pyruvate ketimine . These data demonstrate that quinonoid and ketimine intermediates are catalytically competent in the reaction mechanism of kynureninase, and provide additional support for our proposed mechanism for kynureninase from steady-state kinetic studies {Koushik, S . V., Sundararaju, B., and Phillips, R . S . Biochemistry 1998, 37, 1376-1382}.

Biochim Biophys Acta, 1998 Jun 11, 1385(1), 126 - 38
Purification, molecular characterization and catalytic properties of a Pseudomonas fluorescens enzyme having cholinesterase-like activity; Rochu D et al.; An enzyme with a cholinesterase (ChE) activity, produced by Pseudomonas fluorescens, was purified to homogeneity in a three-step procedure . Analysis by non-denaturing and SDS-PAGE, and by isoelectric focusing, indicated that the enzyme was a monomer of 43 kDa, with a pI of 6.1 . The N-terminal sequence, AEPLKAVGAGEGQLDIVAWPGYIEA, showed some similarities with proteins of the ChE family and a strong similarity with a protein from Escherichia coli with unknown structure and function . Cholinesterase activity at pH 7.0 and 25 degreesC was maximum with propionylthiocholine as substrate (kcat,app=670 min-1), followed by acetylthiocholine, and significantly lower with butyrylthiocholine . Catalytic specificity (kcat/Km) was the same for propionylthiocholine and acetylthiocholine, but was two orders of magnitude lower for butyrylthiocholine . Kinetics of thiocholine ester hydrolysis showed inhibition by excess substrate which was ascribed to binding of a second substrate molecule, leading to non-productive ternary complex (Km=35 microM, KSS=0.49 mM with propionylthiocholine) . There was low or no reactivity with organophosphates and carbamates . The enzyme inhibited by echothiophate (kII=0.44x102 M-1 min-1) was not reactivated by pralidoxime methiodide . However, the P . fluorescens enzyme had affinity for procainamide and decamethonium, two reversible ChE inhibitors used as affinity chromatography ligand and eluant, respectively . Although similarity of the N-terminal amino acid sequence of the enzyme with an internal sequence of ChEs is weak, its catalytic activity towards thiocholine esters, and its affinity for positively charged ligands supports the contention that this enzyme may belong to the ChE family . However, we cannot rule out that the enzyme belongs to another structural family of proteins having cholinesterase-like properties . The reaction of the enzyme with organophosphates suggests that it is a serine esterase, and currently this enzyme may be termed as having a cholinesterase-like activity .

J Dairy Res, 1998 May, 65(2), 261 - 72
Mechanism and kinetics of inactivation at 40-70 degrees C of the extracellular proteinase from Pseudomonas fluorescens 22F; Schokker EP et al.; HPLC size exclusion chromatography experiments showed that during inactivation at 40-70 degrees C of the extracellular proteinase from Pseudomonas fluorescens 22F small molecular mass fragments were formed, indicating that autoproteolysis was at least one of the major causes of inactivation . The formation of small molecular mass fragments and the reaction order indicated that intermolecular autoproteolysis was more likely than intramolecular autodigestion . This was confirmed by computer simulations . The rate constants and the activation enthalpy (delta H++) and entropy (delta S++) for the reactions of the intermolecular autoproteolysis model were derived from computer simulations . delta H++ and delta S++ of the unfolding reaction were 504 kJ mol-1 and 1252 J mol K-1 respectively . delta H++ and delta S++ of the refolding reaction were strongly temperature dependent . The estimates for the enthalpy (delta H0) and entropy (delta S0) difference between the folded and unfolded state as derived from the reaction rate constants of unfolding and refolding were subject to large deviations, owing to accumulation of errors in the estimation of the kinetic characteristics.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7051 - 6
A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365; Dekkers LC et al.; A colonization mutant of the efficient root-colonizing biocontrol strain Pseudomonas fluorescens WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation . The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions . A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to lppL, lysA, dapF, orf235/233, xerC/sss, and the largely incomplete orf238 . The transposon insertion in PCL1233 appeared to be present in the orf235/233 homologue, designated orf240 . Introduction of a mutation in the xerC/sss homologue revealed that the xerC/sss gene homologue rather than orf240 is crucial for colonization . xerC in Escherichia coli and sss in Pseudomonas aeruginosa encode proteins that belong to the lambda integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements . The function of the xerC/sss homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats . Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere . Thus we show the importance of phase variation in microbe-plant interactions.

J Bacteriol, 1998 Jun, 180(12), 3187 - 96
Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0; Laville J et al.; The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions . The genetic basis of HCN synthesis in P . fluorescens CHA0 was investigated . The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium . Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC) . These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2 . The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC .. . ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT .. . ATCAA) . The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P . fluorescens CHA0 and sequenced . ANR of strain CHA0 was most similar to ANR of P . aeruginosa and CydR of Azotobacter vinelandii . An anr mutant of P . fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion . Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions . This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0 . These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

Microbiology, 1998 May, 144 ( Pt 5), 1397 - 405
Metabolism of ferulic acid via vanillin using a novel CoA-dependent pathway in a newly-isolated strain of Pseudomonas fluorescens; Narbad A et al.; A soil bacterium, designated Pseudomonas fluorescens AN103, was isolated based on its ability to grow on ferulic acid as a sole source of carbon and energy . In addition, this strain was found to metabolize a number of related phenolic substrates which contained a hydroxyl group at the para position of the aromatic ring . During growth on ferulic acid, transient accumulation of vanillic acid and trace amounts of protocatechuic acid were detected in the culture medium . Washed cells grown on ferulic acid readily oxidized vanillin, vanillic acid and protocatechuic acid, the three putative intermediates of the metabolic pathway . The side-chain cleavage of ferulic acid to produce vanillin was demonstrated in vitro for the first time and this enzyme-catalysed reaction was shown to have an essential requirement for CoASH, ATP and MgCl2 . This conversion involved a two-step process involving a CoA ligase followed by the side-chain cleavage . The addition of NAD increased the oxidation of vanillin to vanillic acid and had an overall effect of increasing the rate of ferulic acid cleavage . The application of 13C-NMR studies in vitro revealed acetyl-CoA as the C2 side-chain cleavage product . High levels of inducible ferulate-CoA ligase and NAD-linked vanillin dehydrogenase were detected and a novel pathway for ferulic acid metabolism in this organism is proposed.

Curr Microbiol, 1998 Jun, 36(6), 341 - 7
The effect of the lacY gene on the induction of IPTG inducible promoters, studied in Escherichia coli and Pseudomonas fluorescens; Hansen LH et al.; The role of the Escherichia coli lacY gene product (the lactose permease) in the induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible promoters was studied in E . coli and P . fluorescens . This was done by comparing strains containing a lacIPOZYA chromosomal insert with newly constructed strains containing inserts without the lacY gene (lacIPOZ) . The lactose operon inserts were introduced as single-copy chromosomal inserts to eliminate differences in expression caused by differences in copy number . Comparison between the two types of inserts showed that the lactose permease was essential to allow growth on lactose by both bacteria and that the lactose permease plays an important role in transporting the inducer IPTG across the membrane of P . fluorescens . The use of a functional lactose permease allows expression of beta-galactosidase to increase more than fivefold from a wild-type lac promoter in P . fluorescens SS1001 . We suggest that an increase in the rate of protein synthesis from lac-type promoters could be enhanced if an active lactose permease is present as well.

J Dairy Sci, 1998 Apr, 81(4), 901 - 8
Effect of psychrotrophic microorganisms on the plasmin system in milk; Fajardo-Lira CE et al.; The psychrotrophic bacteria that are present in raw milk produce heat-stable proteases that affect the plasmin system and, in turn, the quality of the processed milk and other dairy products . Three bacterial strains, Pseudomonas spp . SRM21A and SRM28A and Pseudomonas fluorescens M3/6, were inoculated at a level of approximately 10(3) cfu/ml into reconstituted nonfat dry milk and incubated at 4 degrees C for up to 9 d . From each sample, casein and whey fractions were obtained and electrophoresed to visualize protein hydrolysis and plasmin activity . Colorimetric assays were used to quantify plasmin-related activities . The casein SDS-PAGE gels showed that, by 5 d of incubation, caseinolytic activity was visible in whey fractions of all three strains at the same molecular mass range as commercial bovine plasmin, which was used as a control . Colorimetric assays indicated that plasmin activity in the casein samples decreased as incubation time increased; plasmin activity in the whey samples increased, peaking at 5 to 7 d . Plasminogen activity decreased over time in the casein fraction and increased in the whey fraction . Protein immunodetection indicated crossreactivity between anti-bovine plasminogen and the plasmin and plasminogen from whey samples collected at 5 and 7 d from the incubated milk . The growth of the Pseudomonas strains tested and their concomitant production of proteases caused the release of plasmin and plasminogen from the casein micelle into the whey fraction.

Appl Microbiol Biotechnol, 1998 Mar, 49(3), 337 - 42
Recalcitrance of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene to degradation by pure cultures of 1,1-diphenylethylene-degrading aerobic bacteria; Megharaj M et al.; 1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) is the peri-chlorinated derivative of 1,1-diphenylethylene (DPE) . Biodegradation of DDE and DPE by bacteria has so far not been shown . Pure cultures of aerobic bacteria involved in biodegradation of styrene and polychlorinated biphenyls (PCB) were therefore screened for their ability to degrade or cometabolize DPE and DDE . Styrene-metabolizing bacteria (Rho-dococcus strains S5 and VLB150) grew with DPE as their sole source of carbon and energy . Polychlorinated-biphenyl-degrading bacteria (Pseudomonas fluorescens and Rhodococcus globerulus) were unable to degrade DPE even in the presence of an easily utilizable cosubstrate, biphenyl . This is the first report of the utilization of DPE as sole carbon and energy source by bacteria . All the tested bacteria failed to degrade DDE when it was provided as the sole carbon source or in the presence of the respective degradable cosubstrates . DPE transformation could also be detected in cell-free extracts of Rhodococcus S5 and VLB150, but DDE was not transformed, indicating that cell wall and membrane diffusion barriers were not limiting biodegradation . The results of the present study show that, at least for some bacteria, the chlorination of DDE is the main reason for its resistance to biodegradation by styrene and DPE-degrading bacteria.

Appl Environ Microbiol, 1998 Apr, 64(4), 1472 - 6
Fluorescent pseudomonad pyoverdines bind and oxidize ferrous ion; Xiao R et al.; Major pyoverdines from Pseudomonas fluorescens 2-79 (Pf-B), P . aeruginosa ATCC 15692 (Pa-C), and P . putida ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand . At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did . Also, increased absorbance at 460 nm was observed to be much faster for Fe2+ -pyoverdine than for Fe3+ -pyoverdine . At pH 7.4, about 90% of Fe3+ was bound by pyoverdine Pa-C after 24 h whereas Fe2+ was bound by the pyoverdine completely in only 5 min . The possibility that Fe2+ underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe2+ concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4 . Incubating excess Fe2+ with pyoverdine in the presence of 8-hydroxyquinoline, an Fe3+ -specific chelating agent, resulted in the formation of a Fe3+ -hydroxyquinoline complex, suggesting that the iron in the Fe2+ -pyoverdine complex existed in the oxidized form . These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.

J Bacteriol, 1998 May, 180(9), 2541 - 8
A seven-gene locus for synthesis of phenazine-1-carboxylic acid by Pseudomonas fluorescens 2-79; Mavrodi DV et al.; Pseudomonas fluorescens 2-79 produces the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA), which is active against a variety of fungal root pathogens . In this study, seven genes designated phzABCDEFG that are sufficient for synthesis of PCA were localized within a 6.8-kb BglII-XbaI fragment from the phenazine biosynthesis locus of strain 2-79 . Polypeptides corresponding to all phz genes were identified by analysis of recombinant plasmids in a T7 promoter/polymerase expression system . Products of the phzC, phzD, and phzE genes have similarities to enzymes of shikimic acid and chorismic acid metabolism and, together with PhzF, are absolutely necessary for PCA production . PhzG is similar to pyridoxamine-5'-phosphate oxidases and probably is a source of cofactor for the PCA-synthesizing enzyme(s) . Products of the phzA and phzB genes are highly homologous to each other and may be involved in stabilization of a putative PCA-synthesizing multienzyme complex . Two new genes, phzX and phzY, that are homologous to phzA and phzB, respectively, were cloned and sequenced from P . aureofaciens 30-84, which produces PCA, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine . Based on functional analysis of the phz genes from strains 2-79 and 30-84, we postulate that different species of fluorescent pseudomonads have similar genetic systems that confer the ability to synthesize PCA.

J Biotechnol, 1998 Feb 5, 60(1-2), 105 - 11
Enantioselectivity of a recombinant esterase from Pseudomonas fluorescens towards alcohols and carboxylic acids; Krebsfanger N et al.; A recombinant esterase from Pseudomonas fluorescens (PFE) was produced from E . coli cultures and the enantioselectivity towards a series of racemic substrates was investigated . PFE exhibited high rate and enantioselectivity in the acylation of alpha-phenyl ethanol with vinyl acetate in toluene (E > 100) and the hydrolysis of the corresponding acetate in phosphate buffer (E = 58) . In sharp contrast, extremely low enantioselectivity (E from 1.1 to 7) was found for the acylation of a series of 1,2-O-protected glycerol derivatives and the hydrolysis of 3-phenylbutyric acid methylester . Almost no reaction occurred with alpha-phenyl propanol and its acetate and 2-phenylbutyric acid ethylester.

J Dairy Sci, 1998 Mar, 81(3), 664 - 71
Effects of hydrolysis of milk glycerides on the antimutagenicity of a hexane extract of milk; Nadathur SR et al.; Reconstituted nonfat dry milk was treated with different amounts of lipase from Pseudomonas fluorescens . Hexane extracts of treated milks were dissolved in dimethylsulfoxide and assayed for antimutagenicity using the Ames test (Salmonella typhimurium TA 100) against N-methyl, N'-nitro, N-nitrosoguanidine . Anti-N-methyl, N'-nitro, N-nitrosoguanidine activity increased significantly as the amount of added lipase increased . At the highest lipase concentration tested, activity increased 5-fold, suggesting that liberated fatty acids contributed to the increased antimutagenicity . The activities of mixtures of pure fatty acids on antimutagenesis were examined using the Ames test . At the lowest concentrations tested, mixtures of palmitic and stearic acids and mixtures of palmitic and isopalmitic acids exhibited greater activity than did the individual acids . At all doses tested, mixtures of the monoacylglycerides of palmitic and stearic acids exhibited the same activity as the individual components . Quantification of fatty acids in milk and yogurt by gas chromatography indicated a 2 to 20-fold greater content of free fatty acids in yogurt . The increase in free fatty acids may contribute to the increase in antimutagenicity of yogurt relative to that of milk.

Int J Food Microbiol, 1998 Jan 6, 39(1-2), 53 - 60
Growth of Pseudomonas fluorescens and Pseudomonas fragi in a meat medium as affected by pH (5.8-7.0), water activity (0.97-1.00) and temperature (7-25 degrees C); Lebert I et al.; A total of 59 strains of Pseudomonas, isolated from meat products, were grown in micro-titer plates in a meat medium over a range of pH (5.8-7.0), alpha w (0.97-1.00) and temperature (7-25 degrees C) . Growths were performed in a meat broth with an automated turbidimeter (Bioscreen C, Labsystem, France) . The growth curves obtained in this study did not have sigmoidal shapes making it impossible to calculate growth parameters using the Gompertz equation . The medium was weakly oxygenated in the micro-titer plates and reached 0%-dissolved oxygen at the beginning of the exponential phase . Strains were separated into two groups: P . fragi and P . fluorescens . Strains of P . fragi had shorter lag times than those of P . fluorescens . The impact of such results is interesting in that these could assist to explain the succession of flora that is observed during the processing of meat: P . fluorescens is the dominant bacteria among Pseudomonas spp . at the beginning of a slaughter line and P . fragi becomes dominant during the chilling process.

Int J Food Microbiol, 1998 Feb 17, 39(3), 185 - 94
Identification of pseudomonads from fresh and chill-stored chicken carcasses; Sundheim G et al.; Results of carbon source assimilation tests (17 carbon compounds) led to 88% of pseudomonads from cold-stored chicken carcasses being assigned to one of 17 groups . Of these groups, 13 had combinations of properties identical to, or with readily recognizable degrees of similarity to those of published species/biovars . Two of the four groups having carbon assimilation patterns dissimilar to any known species had cellular fatty acid composition corresponding to Pseudomonas fluorescens, and two to Pseudomonas lundensis or Pseudomonas fragi . The P . fluorescens biovars all had higher amounts of 16:1 cis 9 (21-37%) and 18:1 cis 11 (10-19%), than of 17:0 cyclo (1-17%) and 19:0 cyclo (0-1%) . In contrast, for P . lundensis and P . fragi, the relative amounts of these unsaturated acids and cyclopropane acids were reversed . Both the carbon source assimilation tests and the cellular fatty acid composition led to the conclusion that none of the species were dominant, although the P . fluorescens biovars constituted about 50% of the isolated pseudomonads.

J Bacteriol, 1998 Apr, 180(7), 1939 - 43
Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens; Kirner S et al.; Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity . Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin . In the work presented here, we describe the function of each prn gene product . The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted . The prnA gene product catalyzes the chlorination of L-tryptophan to form 7-chloro-L-tryptophan . The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-L-tryptophan to monodechloroaminopyrrolnitrin . The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin . The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin . The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.

J Biol Chem, 1998 Feb 13, 273(7), 4163 - 70
Metabolism of ferulic acid to vanillin . A bacterial gene of the enoyl-SCoA hydratase/isomerase superfamily encodes an enzyme for the hydration and cleavage of a hydroxycinnamic acid SCoA thioester; Gasson MJ et al.; A gene encoding a novel enoyl-SCoA hydratase/lyase enzyme for the hydration and nonoxidative cleavage of feruloyl-SCoA to vanillin and acetyl-SCoA was isolated and characterized from a strain of Pseudomonas fluorescens . Feruloyl-SCoA is the CoASH thioester of ferulic acid (4-hydroxy-3-methoxy-trans-cinnamic acid), an abundant constituent of plant cell walls and a degradation product of lignin . The gene was isolated by a combination of mutant complementation and biochemical approaches, and its function was demonstrated by heterologous expression in Escherichia coli under the control of a T7 RNA polymerase promoter . The gene product is a member of the enoyl-SCoA hydratase/isomerase superfamily.

J Med Microbiol, 1998 Mar, 47(3), 197 - 200
Plasmid-mediated rifampicin resistance in Pseudomonas fluorescens; Chandrasekaran S et al.; Rifampicin is an antibiotic mostly used to treat tuberculosis and leprosy, and, occasionally, other diseases . Resistance is due to alterations in membrane permeability or to mutation in the rpoB gene coding for mRNA polymerase . Both these mechanisms originate via chromosomal mutation . However, a rifampicin-resistant Pseudomonas fluorescens strain harboured a multiresistance plasmid which transferred rifampicin resistance when transformed into P . putida or Escherichia coli . Rifampicin readily diffused into the sensitive cells of the E . coli and P . putida recipients, but the transformants with the plasmid, pSCL were resistant to the drug and did not accumulate it . Potassium cyanide restored the diffusion of rifampicin into the resistant cells, indicating that an efflux pump was involved in the resistance mechanism . The resistance of the transformants and the wild strain was also abolished in sphaeroplasts generated by EDTA lysozyme treatment . Analysis of membrane proteins by SDS-PAGE revealed the presence of two new proteins in the plasmid-containing cells of E . coli, P . putida and P . fluorescens and not in the plasmid-free cells . These may be involved in the efflux of rifampicin.

J Bacteriol, 1998 Mar, 180(5), 1063 - 71
Genetic and functional analysis of the styrene catabolic cluster of Pseudomonas sp . strain Y2; Velasco A et al.; The chromosomal region of Pseudomonas sp . strain Y2 involved in the conversion of styrene to phenylacetate (upper catabolic pathway) has been cloned and sequenced . Four catabolic genes, styABCD, and two regulatory genes, stySR, were identified . This gene cluster when transferred to Escherichia coli W confers to this phenylacetate-degrading host the ability to grow on styrene as the sole carbon and energy source . Genes styABCD are homologous to those encoding the styrene upper catabolic pathway in Pseudomonas fluorescens ST . Northern blot analyses have confirmed that genes styABCD constitute a transcription unit . The transcription start site of the sty operon was mapped 33 nucleotides upstream of the styA translational start codon . The styS and styR genes, which form an independent transcriptional unit, are located upstream of the styABCD operon, and their gene products show high similarity to members of the superfamily of two-component signal transduction systems . The styS gene product is homologous to histidine kinase proteins, whereas the styR gene product exhibits similarity at its N-terminal domain with cluster 1 of receiver modules and at its C terminus with the LuxR/FixJ family 3 of DNA-binding domains . Expression of the catabolic operon decreased significantly in the absence of the stySR genes and was restored when the stySR genes were provided in trans in the presence of styrene, suggesting that the stySR system behaves as a styrene-inducible positive regulator of the styABCD operon . Finally, a gene encoding a phenylacetyl-coenzyme A ligase that catalyzes the first step in the phenylacetate catabolism (styrene lower catabolic pathway) has been identified upstream of the styS gene . This activity was found to be induced in Pseudomonas sp . strain Y2 cells grown on styrene but not present in cells grown on glycerol . These results strongly suggest that the genes responsible for the complete mineralization of styrene are clustered in the chromosome of Pseudomonas sp . strain Y2.

Biochemistry, 1998 Feb 3, 37(5), 1376 - 82
The catalytic mechanism of kynureninase from Pseudomonas fluorescens: insights from the effects of pH and isotopic substitution on steady-state and pre-steady-state kinetics; Koushik SV et al.; The effects of pH and isotopic substitution of substrate and solvent on the reaction of kynureninase from Pseudomonas fluorescens have been determined . The pH dependence of kcat/Km for L-kynurenine is bell-shaped, with apparent pKa's of 6.25 +/- 0.05 on the acidic limb and 8.9 +/- 0.1 on the basic limb, and with a pH-dependent value of kcat/Km of 2 x 10(5) M-1 s-1 . The pH dependence of kcat/Km for 3-hydroxykynurenine is also bell-shaped, with apparent pKa's of 6.49 +/- 0.07 and 8.55 +/- 0.09, and with a pH-dependent value of 2.5 x 10(3) M-1 s-1 . The kcat for L-kynurenine decreases at acidic pH values, with an apparent pKa of 6.43 +/- 0.06 and a pH-dependent value of 7 s-1 . The solvent kinetic isotope effect on kcat for the reaction of kynurenine in {2H}H2O is 6.56 +/- 0.59, whereas there is no normal kinetic isotope effect on kcat/Km, at pH 8.1 . The proton inventory of kcat fits very well to the Gross-Butler equation, with x = 0.825 +/- 0.08, suggesting that only a single proton is transferred in the rate-determining step . In contrast, there is no significant kinetic isotope effect on either kcat or kcat/Km with alpha-{2H}-L-kynurenine as the substrate . There is a "burst" of anthranilate (0.7 mol/mol of enzyme) formed in the pre steady state of the reaction of kynureninase, with a rate constant of 54 s-1 which is not affected by {2H}H2O . The partition ratio of alanine to pyruvate formation is 2.3 x 10(4) in H2O and 6.9 x 10(3) in {2H}H2O . Taken together, these data indicate that the rate-limiting step in the reaction of kynureninase occurs subsequent to the first irreversible step, which is anthranilate release, is general base catalyzed, and involves transfer of only a single proton . On the basis of these observations, we propose that the rate-limiting step in the reaction of kynureninase is C-4' deprotonation of the pyruvate pyridoxamine 5'-phosphate ketimine intermediate.

Gene, 1998 Jan 5, 206(1), 117 - 26
Structure and function of the genes involved in mannitol, arabitol and glucitol utilization from Pseudomonas fluorescens DSM50106; Brunker P et al.; A DNA fragment from Pseudomonas fluorescens DSM50106 containing the genes for the uptake and utilization of mannitol, arabitol and glucitol was cloned in Escherichia coli and sequenced . Seven open reading frames (mtlEFGKDYZ) were identified on the 10031 bp fragment . The deduced amino acid sequences of the first four open reading frames (mtlEFGK) revealed significant similarity to the components of the maltose transport system in E . coli and Salmonella typhimurium . The gene mtlD encoding a polyol dehydrogenase was located downstream of mtlK . The deduced proteins of the last two genes on the fragment showed a high similarity to a fructokinase from Vibrio alginolyticus (MtlZ) and a xylulose kinase from Streptomyces rubiginosus (MtlY), respectively . Both genes were expressed in E . coli . MtlZ phosphorylated fructose, glucose and glucitol whereas MtlY was highly specific for xylulose . Upstream of mtlE, a putative promoter/operator region was identified by promoter probe studies which was active in P . fluorescens but not in E . coli.

Protein Sci, 1997 Nov, 6(11), 2454 - 8
Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding; Eppink MH et al.; A novel conserved sequence motif has been located among the flavoprotein hydroxylases . Based on the crystal structure and site-directed mutagenesis studies of p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, this amino acid fingerprint sequence is proposed to play a dual function in both FAD and NAD(P)H binding . In PHBH, the novel sequence motif (residues 153-166) includes strand A4 and the N-terminal part of helix H7 . The conserved amino acids Asp 159, Gly 160, and Arg 166 are necessary for maintaining the structure . The backbone oxygen of Cys 158 and backbone nitrogens of Gly 160 and Phe 161 interact indirectly with the pyrophosphate moiety of FAD, whereas it is known from mutagenesis studies that the side chain of the moderately conserved His 162 is involved in NADPH binding.

Structure, 1997 Dec 15, 5(12), 1571 - 84
Crystal structure of carboxylesterase from Pseudomonas fluorescens, an alpha/beta hydrolase with broad substrate specificity; Kim KK et al.; BACKGROUND: A group of esterases, classified as carboxylesterases, hydrolyze carboxylic ester bonds with relatively broad substrate specificity and are useful for stereospecific synthesis and hydrolysis of esters . One such carboxylesterase from Pseudomonas fluorescens is a homodimeric enzyme, consisting of 218-residue subunits . It shows a limited sequence similarity to some members of the alpha/beta hydrolase superfamily . Although crystal structures of a number of serine esterases and lipases have been reported, structural information on carboxylesterases is very limited . This study was undertaken in order to provide such information and to understand a structural basis for the substrate specificity of this carboxylesterase . RESULTS: In this study, the crystal structure of carboxylesterase from P . fluorescens has been determined by the isomorphous replacement method and refined to 1.8 A resolution . Each subunit consists of a central seven-stranded beta sheet flanked by six alpha helices . The structure reveals the catalytic triad as Ser 114-His 199-Asp 168 . The structure of the enzyme in complex with the inhibitor phenylmethylsulfonyl fluoride has also been determined and refined to 2.5 . The inhibitor is covalently attached to Ser 114 of both subunits, with the aromatic ring occupying a hydrophobic site defined by the aliphatic sidechains of Leu23, Ile58, Ile70, Met73 and Val170 . No large structural changes are observed between the free and inhibitor-bound structures . CONCLUSIONS: Carboxylesterase from P . fluorescens has the alpha/beta hydrolase fold and the Ser-His-Asp catalytic triad . The active-site cleft in each subunit is formed by the six loops covering the catalytic serine residue . Three of the active-site loops in each subunit are involved in a head-to-head subunit interaction to form a dimer; it may be these extra structural elements, not seen in other esterases, that account for the inability of carboxylesterase to hydrolyze long chain fatty acids . As a result of dimerization, the active-site clefts from the two subunits merge to form holes in the dimer . The active-site clefts are relatively open and thus the catalytic residues are exposed to the solvent . An oxyanion hole, formed by nitrogen atoms of Leu23 and Gln115, is present in both the free and inhibitor-bound structures . An open active site, as well as a large binding pocket for the acid part of substrates, in P . fluorescens carboxylesterase may contribute to its relatively broad substrate specificity.

J Ind Microbiol Biotechnol, 1997 Nov-Dec, 19(5-6), 378 - 84
Microbial models of soil metabolism: biotransformations of danofloxacin; Chen Y et al.; Danofloxacin is a new synthetic fluoroquinolone antibacterial agent under development for exclusive use in veterinary medicine . Such use could lead to deposition of low levels of danofloxacin residues in the environment in manure from treated livestock . This study was conducted to evaluate the potential for indigenous soil microorganisms to metabolize danofloxacin . Cultures of 72 soil microorganisms representing a diverse panel of bacteria, fungi and yeast were incubated with danofloxacin mesylate substrate and samples analyzed periodically by high performance liquid chromatography for loss of danofloxacin and formation of metabolites . Some samples were further analyzed by liquid chromatography-mass spectrometry and mass spectrometry to confirm metabolite identification . Twelve organisms, representing eight different genera, biotransformed danofloxacin to metabolites detectable by the chromatographic methods employed . Two Mycobacterium species, two Pseudomonas species, and isolates of Nocardia sp, Rhizopus arrhizus and Streptomyces griseus all formed N-desmethyldanofloxacin . The formation of the 7-amino danofloxacin derivative, 1-cyclopropyl-6-fluoro-7-amino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid by cultures of Candida lipopytica, Pseudomonas fluorescens, two Mycobacterium species and three Penicillium species demonstrates the propensities of these cultures to completely degrade the piperazine ring . At least two additional and unidentified metabolite peaks were observed in chromatograms of Aspergillus nidulans and Penicillium sp cultures . Radiolabled {2-14C}danofloxacin added to cultures of the fungus Curvularia lunata was apparently mineralized, with approximately 31% of the radiolabel recovered as volatile metabolites after 24 h of incubation, indicating the susceptibility of the quinolone ring to microbial metabolic degradation.

Folia Microbiol (Praha), 1997, 42(4), 345 - 8
Lipase activity of Pseudomonas fluorescens in cold raw skim milk with different lipid supplements; Perez-Esteban J et al.; Different dairy-like fat fractions were added to raw skim milk in order to determine their effect on lipase production by Pseudomonas fluorescens growing on raw milk at 7 degrees C . Supplementation with preparations of fat globule membranes, mixtures of capric acid, glycerol monocaprinate, and glycerol dicaprinate, cholesterol, or lecithin at concentrations similar to those occurring in whole milk had only slight effects on lipase production, whereas supplementation with 0.9% glycerol trioleate decreased lipase activity in milk by 90%.

Antonie Van Leeuwenhoek, 1997 Nov, 72(4), 365 - 72
Use of a lux-based procedure to rapidly visualize root colonisation by Pseudomonas fluorescens in the wheat rhizosphere; de Weger LA et al.; The bioluminescently marked Pseudomonas fluorescens strain 5RL, has been used previously to follow colonisation of soy bean roots (De Weger et al . {1991} Appl . Environ . Microbiol . 57:36-41) . In the present paper the method has been further developed and optimized for wheat roots and it is used to get a quick overview of the colonisation patterns of many different root systems at the same time . Colonisation was followed on wheat plants grown in our gnotobiotic sand system (Simons et al., 1996 . Mol Plant Microbe Interact 9: 600-607) and the following results were obtained . (i) A spatio-temporal analysis of the colonisation of wheat roots showed that 4 days after planting the highest bacterial activity was observed at the upper part of the root . After 6 days the high bacterial activity at the upper part was further increased, whereas spot-like activities were observed on the lower root parts, possibly due to micro-colonies . (ii) Bacterial mutations causing lack of motility or auxotrophy for amino acids resulted in impaired colonisation of the lower root parts, indicating that motility and prototrophy for the involved amino acid(s) are important factors for wheat root colonisation by strain 5RL . (iii) Coinoculation of strain 5RL with other wild type Pseudomonas strains on the root influenced the colonisation pattern observed for strain 5RL . Colonisation was not visually affected when the competing strain was a poor root coloniser, but was severely reduced when the competing strain was a good root coloniser . The results show that the spatio-temporal colonisation of wheat root by P . fluorescens strain 5RL and derivatives is similar to that of strain WCS365 on tomato . The advantage of the use of lux-marked strains is that the results are obtained much quicker than when conventional methods are used and that the result is supplied as an image of the colonisation pattern of many different roots.

Gene, 1997 Dec 19, 204(1-2), 17 - 24
Identification and sequence analysis of the genes encoding a polyketide synthase required for pyoluteorin biosynthesis in Pseudomonas fluorescens Pf-5; Nowak-Thompson B et al.; Pyoluteorin is a chlorinated antifungal metabolite of mixed polyketide/amino-acid origin produced by certain strains of Pseudomonas spp., including the soil bacterium Pseudomonas fluorescens Pf-5 . Sequence analysis of a gene cluster required for pyoluteorin biosynthesis by Pf-5 (Kraus, J., Loper, J., 1995 . Appl . Environ . Microbiol . 61, 849-854) has identified two genes whose deduced peptide sequences exhibit characteristics of both fungal and bacterial Type I polyketide synthases (PKSs) . The pyoluteorin PKS does not contain a loading domain that is typically present in bacterial Type I PKSs . Furthermore, this PKS possesses an acyltransferase domain that does not contain the conserved residues surrounding the active-site motif typically found in domains of similar function . Based on the organization of the functional domains within the pyoluteorin PKS, we propose a biosynthetic pathway analogous to non-aromatic polyketide biosynthesis within the Actinomycete bacteria that is responsible for the formation of the resorcinol moiety of pyoluteorin.

Mol Plant Microbe Interact, 1998 Jan, 11(1), 45 - 56
A two-component system plays an important role in the root-colonizing ability of Pseudomonas fluorescens strain WCS365; Dekkers LC et al.; We describe the characterization of a novel Tn5lacZ colonization mutant of the efficiently colonizing Pseudomonas fluorescens strain WCS365, mutant strain PCL1210, which is at least 300- to 1,000-fold impaired in colonization of the potato root tip after co-inoculation of potato stem cuttings with a 1:1 mixture of mutant and parental cells . Similarly, the mutant is also impaired in colonization of tomato, wheat, and radish, indicating that the gene involved plays a role in the ability of P . fluorescens WCS365 to colonize a wide range of plant species . A 3.1-kb DNA fragment was found to be able to complement the observed mutation . The nucleotide sequence of the region around the Tn5lacZ insertion showed three open reading frames (ORFs) . The transcriptional start site was determined . The operon is preceded by an integration host factor (IHF) binding site consensus sequence whereas no clear -10 and -35 sequences are present . The deduced amino acid sequences of the first two genes of the operon, designated as colR and colS, show strong similarity with known members of two-component regulatory systems . ColR has homology with the response regulators of the OmpR-PhoB subclass whereas ColS, the product of the gene in which the mutation resides, shows similarity to the sensor kinase members of these two-component systems . Hydrophobicity plots show that this hypothetical sensor kinase has two transmembrane domains, as is also known for other sensor kinases . The product of the third ORF, Orf222, shows no homology with known proteins . Only part of the orf222 gene is present in the colonization-complementing, 3.1-kb region, and it therefore does not play a role in complementation . No experimental evidence for a role of the ColR/ColS two-component system in the suspected colonization traits chemotaxis and transport of exudate compounds could be obtained . The function of this novel two-component system therefore remains to be elucidated . We conclude that colonization is an active process in which an environmental stimulus, through this two-component system, activates a so far unknown trait that is crucial for colonization.

Mikrobiologiia, 1997 Sep-Oct, 66(5), 588 - 94
{Toxic effect of hydroxylated ions of heavy metals on the cytoplasmic membrane of bacterial cells}; Ivanov AIu et al.; The influence of nickel, copper, cadmium, and lead ions at concentrations of 50 to 100 microM on the barrier properties of the plasma membrane (PM) and the electrophoretic mobility (EPM) of Pseudomonas fluorescens 71, Escherichia coli K-12, and Mycobacterium phlei B-1291 VKM cells was studied at pH values from 5 to 9 by electro-orientational (EO) spectroscopy and microelectrophoresis of cells . According to the data of EO spectroscopy, the increase in the toxicity of heavy metal cations to cells corresponded to transition of cations to monovalent hydroxylated forms . Hydroxylated ions were found to more easily adsorb on, or penetrate across, the PM and to bind to competent proteins . During the treatment of all three investigated microorganisms with Cu and Pb ions, and gram-negative bacteria also with Ni ions, the EPM of cells changed in a pH range corresponding to the transition of bivalent metal ions to their monovalent hydroxylated forms . Changes in the EPM induced by increasing pH correlated well with the enhanced toxicity of these metals to the PM, as evidenced by the EO spectroscopy data . At the same time, this correlation was less pronounced for cadmium sulfate toxicity to all of the microorganisms studied and for nickel chloride toxicity to M . phlei cells.

Biochemistry, 1997 Dec 9, 36(49), 15489 - 500
Evidence that galactanase A from Pseudomonas fluorescens subspecies cellulosa is a retaining family 53 glycosyl hydrolase in which E161 and E270 are the catalytic residues; Braithwaite KL et al.; A genomic library of Pseudomonas fluorescens subsp . cellulosa DNA was screened for galactanase-positive recombinants . The nine galactanase positive phage isolated contained the same galactanase gene designated galA . The deduced primary structure of the enzyme (galactanase A; GalA) encoded by galA had a Mr of 42 130 and exhibited significant sequence identity with a galactanase from Aspergillus aculeatus, placing GalA in glycosyl hydrolase family 53 . The enzyme displayed properties typical of an endo-beta1, 4-galactanase and exhibited no activity against the other plant structural polysaccharides evaluated . Analysis of the stereochemical course of 2,4-dinitrophenyl-beta-galactobioside (2,4-DNPG2) hydrolysis by GalA indicated that the galactanase catalyzes the hydrolysis of glycosidic bonds by a double displacement general acid-base mechanism . Hydrophobic cluster analysis (HCA) suggested that family 53 enzymes are related to the GH-A clan of glycosyl hydrolases, which have an (alpha/beta)8 barrel structure . HCA also predicted that E161 and E270 were the acid-base and nucleophilic residues, respectively . Mutants of GalA in which E161 and E270 had been replaced with alanine residues were essentially inactive against galactan . Against 2,4-DNPG2, E161A exhibited a much lower Km and kcat than native GalA, while E270A was inactive against the substrate . Analysis of the pre-steady-state kinetics of 2,4-DNPG2 hydrolysis by E161A showed that there was an initial rapid release of 2,4-dinitrophenol (2,4-DNP), which then decayed to a slow steady-state rate of product formation . No pre-steady-state burst of 2,4-DNP release was observed with the wild-type enzyme . These data are consistent with the HCA prediction that E161 and E270 are the acid-base and nucleophilic catalytic residues of GalA, respectively.

J Biol Chem, 1997 Nov 28, 272(48), 30254 - 60
Specificity of substrate recognition by Pseudomonas fluorescens N3 dioxygenase . The role of the oxidation potential and molecular geometry; Di Gennaro P et al.; Pseudomonas fluorescens N3 is able to grow on naphthalene as the sole carbon and energy source . The mutant TTC1, blocked at the dihydrodiol dehydrogenase level, which can transform the hydrocarbon into the corresponding dihydrodiol, has been used to produce bioconversion products . To rationalize the different grades of conversion obtained with different substrates, a study was performed using non-naphthalene derivatives, including benzenes, conjugated benzenes, and polycyclic aromatic hydrocarbons . The corresponding diols obtained by bioconversion have been isolated and characterized . A theoretical model that considers both energy and geometry factors has been proposed to rationalize the experimental data . Good agreement has been found between the calculated values and the experimental results.

Appl Environ Microbiol, 1997 Dec, 63(12), 4920 - 8
Distribution of metabolic activity and phosphate starvation response of lux-tagged Pseudomonas fluorescens reporter bacteria in the barley rhizosphere; Kragelund L et al.; The purpose of this study was to determine the metabolic activity of Pseudomonas fluorescens DF57 in the barley rhizosphere and to assess whether sufficient phosphate was available to the bacterium . Hence, two DF57 reporter strains carrying chromosomal luxAB gene fusions were introduced into the rhizosphere . Strain DF57-40E7 expressed luxAB constitutively, making bioluminescence dependent upon the metabolic activity of the cells under defined assay conditions . The DF57-P2 reporter strain responded to phosphate limitation, and the luxAB gene fusion was controlled by a promoter containing regulatory sequences characteristic of members of the phosphate (Pho) regulon . DF57 generally had higher metabolic activity in a gnotobiotic rhizosphere than in the corresponding bulk soil . Within the rhizosphere the distribution of metabolic activity along the root differed between the rhizosphere soil and the rhizoplane, suggesting that growth conditions may differ between these two habitats . The DF57-P2 reporter strain encountered phosphate limitation in a gnotobiotic rhizosphere but not in a natural rhizosphere . This difference in phosphate availability seemed to be due to the indigenous microbial population, as DF57-P2 did not report phosphate limitation when established in the rhizosphere of plants in sterilized soil amended with indigenous microorganisms.

Appl Environ Microbiol, 1997 Dec, 63(12), 4793 - 9
Partial purification and characterization of manganese-oxidizing factors of Pseudomonas fluorescens GB-1; Okazaki M et al.; The Mn(2+)-oxidizing bacterium Pseudomonas fluorescens GB-1 deposits Mn oxide around the cell . During growth of a culture, the Mn(2+)-oxidizing activity of the cells first appeared in the early stationary growth phase . It depended on the O2 concentration in the culture during the late logarithmic growth phase . Maximal activity was observed at an oxygen concentration of 26% saturation . The activity could be recovered in cell extracts and was proportional to the protein concentration in the cell extracts . The specific activity was increased 125-fold by ammonium sulfate precipitation followed by reversed-phase and gel filtration column chromatographies . The activity of the partly purified Mn(2+)-oxidizing preparation had a pH optimum of circa 7 and a temperature optimum of 35 degrees C and was lost by heating . The Mn(2+)-oxidizing activity was sensitive to NaN3 and HgCl2 . It was inhibited by KCN, EDTA, Tris, and o-phenanthroline . Although most data indicated the involvement of protein in Mn2+ oxidation, the activity was slightly stimulated by sodium dodecyl sulfate at a low concentration and by treatment with pronase and V8 protease . By polyacrylamide gel electrophoresis, two Mn(2+)-oxidizing factors with estimated molecular weights of 180,000 and 250,000 were detected.

Appl Environ Microbiol, 1997 Nov, 63(11), 4340 - 5
Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400; Gaballa A et al.; Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation . A lacZ reporter gene introduced by transposon mutagenesis into the P . fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum . The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase . A trehalose concentration as low as 1 microM could induce the expression of treA . The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P . fluorescens ATCC 17400 to utilize trehalose as a carbon source . A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene . This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation . These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism.

Appl Environ Microbiol, 1997 Nov, 63(11), 4261 - 6
Evidence for signaling between the phytopathogenic fungus Pythium ultimum and Pseudomonas fluorescens F113: P . ultimum represses the expression of genes in P . fluorescens F113, resulting in altered ecological fitness; Fedi S et al.; There is increasing evidence that communication between members of the same species, as well as members of different species, is important for the survival of microorganisms in diverse ecological niches, such as the rhizosphere . To investigate whether the phytopathogen Pythium ultimum could alter gene expression in the biocontrol strain Pseudomonas fluorescens F113, which protects the roots of sugar beet from the fungus, a screening system was developed to detect differential expression of bacterial genes in the presence of P . ultimum . The transposon Tn5, containing a promoterless lacZ reporter gene, was used to generate a library of transcriptional gene fusions in P . fluorescens F113 . By this screening procedure, five P . fluorescens F113 gene clusters were identified and shown to be repressed in the presence of P . ultimum . The ecological fitness of three of the five reporter mutants in the rhizosphere of seed-inoculated sugar beet was lower than that of the wild type . Furthermore, all five mutants were impaired in their ability to subsequently colonize the rhizosphere of uninoculated sugar beet sown repeatedly in the same soil . With the exception of reporter mutant SF10, which was impaired in nitrogen metabolism, the reporter mutants had growth requirements and biocontrol abilities similar to those of the wild type . This is the first reported case of a fungus repressing the expressing of bacterial genes.

J Dairy Sci, 1997 Oct, 80(10), 2297 - 303
Effect of high hydrostatic pressure on Escherichia coli and Pseudomonas fluorescens strains in ovine milk; Gervilla R et al.; Ovine milk that had been standardized to 6% fat was inoculated with Escherichia coli 405 CECT and Pseudomonas fluorescens 378 CECT at a rate of 10(6) and 10(7) cfu/ml, respectively, and treated with high hydrostatic pressure . Treatments consisted of combinations of pressure (300, 400, 450, and 500 MPa), temperature (2, 10, 25, and 50 degrees C), and time (5, 10, and 15 min) . Inactivation (> 6 log cfu/ml) of both strains was observed at 50 degrees C for all pressures and treatment times . A similar level of inactivation occurred at > or = 450 MPa and 25 degrees C for E . coli and at > or = 400 MPa and 10 degrees C for P . fluorescens . Destruction was lowest at 10 degrees C for E . coli and at 25 degrees C for P . fluorescens . The test strain of E . coli was more baroresistance than was the P . fluorescens strain.

Chemosphere, 1997 Nov, 35(9), 1967 - 85
Toxicity assessment of xenobiotic contaminated groundwater using lux modified Pseudomonas fluorescens; Boyd EM et al.; A bacterial bioassay, suitable for rapid screening to assess the relative toxicity of xenobiotic contaminated groundwater has been developed . The quantitative bioassay utilizes a decline in luminescence of the lux marked soil bacterium Pseudomonas fluorescens on exposure to contaminated groundwaters from which effective concentration (EC) values can be assessed and compared . P . fluorescens was most sensitive to semivolatile organics in groundwaters but there was no correlation between EC value and chemical content . The sensitivity and reproducibility of the P . fluorescens bioassay was compared with that of Microtox and results showed that mean EC50 values for diluted ground water replicate samples were 20% and 18% respectively . This suggested that the P . fluorescens bioassay was as applicable to groundwater screening as the widely used Microtox bioassay.

FEMS Microbiol Lett, 1997 Oct 15, 155(2), 209 - 15
Pyroglutamic acid and iron regulate the expression of the pcp gene in Pseudomonas fluorescens MFO; Le Saux O et al.; Pyrrolidone carboxyl peptidase (Pcp) is an aminopeptidase (EC 3.4.11.8) able to specifically remove the L-pyroglutamyl residue from the amino-terminus of polypeptides . Since nothing was known concerning the regulation and function of Pcps, a mutant of a milk-isolated strain lacking Pcp activity (Pseudomonas fluorescens MB1), was constructed by homologous recombination using a transcriptional fusion between pcp and a reporter gene (uidA) . The wild-type and mutant strains were grown in synthetic media and in milk to investigate the environmental effects on pcp transcription . The expression of pcp and of the transcriptional fusion pcp::uidA was not sensitive to environmental conditions like temperature, osmolarity or nitrogen and phosphate starvation but was induced by the product of the enzymatic activity, pyroglutamic acid (pGlu) . The expression of the native gene and the fusion in inducing conditions was also controlled by the iron concentration . The identification in the pcp promoter sequence of putative ferric uptake regulator (Fur) binding sites suggests a transcriptional regulation in a Fur-dependent fashion . Two other putative regulatory stretches, corresponding to inverted repeated sequences with perfect and imperfect symmetry, were also identified . pGlu and iron are therefore at least two of the transcriptional effectors of pcp expression.

Biochem Biophys Res Commun, 1997 Oct 20, 239(2), 393 - 400
Nucleotide sequence and genetic complementation analysis of lep from Azotobacter vinelandii; Jock CA et al.; The lep of Azotobacter vinelandii is an 852-base-pair open reading frame (ORF) which encodes a protein of 284 amino acid residues . The translated protein shares 75% homology with leader peptidase I isolated from Pseudomonas fluorescens and 37% homology with leader peptidase I isolated from Escherichia coli . Five highly conserved regions found in the family of leader peptidase I proteins are conserved in A . vinelandii Lep . The putative membrane topology of the protein seems similar to that of E . coli leader peptidase I based on the hydrophobicity analysis of the predicted amino acid sequence . Southern blotting analysis of the A . vinelandii chromosome by probing with lep specific DNA revealed that lep is present as a single copy per the chromosome . A multicopy plasmid carrying A . vinelandii lep could complement a temperature sensitive lep mutant of E . coli strain IT41, suggesting that we have identified the functional copy of lep present on A . vinelandii genome .

Mol Gen Genet, 1997 Sep, 256(1), 84 - 7
Single-step conjugative cloning of bacterial gene fusions involved in microbe-host interactions; Rainey PB et al.; In vivo expression technology (IVET) is a genetic strategy for isolating genes expressed in vivo . In order to full exploit this technology, it is necessary to analyse large numbers of IVET-generated gene fusions, which must be recovered from the chromosome of host bacteria . In bacteria for which transductional methods are not available, the recovery of integrated fusion plasmids is problematic and currently limits broad application of IVET . We describe a rapid, single-step, triparental conjugative approach for recovering chromosomally integrated fusion plasmids from both Pseudomonas fluorescens and Salmonella typhimurium . This simple and broadly applicable conjugative cloning system extends the utility of the IVET approach to clinically and agronomically relevant microbes and may be employed to recover non-replicating and integrated plasmids in other systems.

Am J Health Syst Pharm, 1997 Oct 1, 54(19), 2185 - 91
Intranasal mupirocin for outbreaks of methicillin-resistant Staphylococcus aureus; Bertino JS Jr; The pharmacology, pharmacokinetics, clinical efficacy, adverse effects, and dosage and administration of mupirocin are reviewed . Mupirocin is a naturally occurring antibiotic produced by submerged fermentation of Pseudomonas fluorescens . It inhibits bacterial protein synthesis by binding reversibly and specifically to isoleucyl-tRNA synthetase . Organisms resistant to other antimicrobials are not simultaneously resistant to mupirocin . Mupirocin is highly active against Staphylococcus aureus and other staphylococci and streptococci . When mupirocin ointment is applied topically, local concentrations exceed the inhibitory concentrations for staphylococci and remain detectable for up to 72 hours . Placebo-controlled studies demonstrate the ability of mupirocin to eliminate nasal carriage of S . aureus in health care workers . Observational studies suggest that mupirocin is efficacious in treating methicillin-resistant S . aureus (MRSA) outbreaks . Preliminary studies show that mupirocin might have a role in preventing infections in high-risk patients . Although mupirocin seems to be well tolerated, mild to moderate adverse events have been reported, including respiratory problems and effects confined to the nose--erythema, swelling, burning or stinging, pruritus, and dryness . Mupirocin calcium ointment has FDA-approved labeling for the eradication of nasal MRSA colonization in adult patients and health care workers as part of comprehensive infection-control programs to reduce the risk of infection during institutional outbreaks . The recommended dosage is 0.5 g inserted into each nostril twice daily for five days . Intranasal mupirocin ointment appears to be a useful addition to infection-control programs designed to reduce the risk of infection among patients during MRSA outbreaks.

Sci Total Environ, 1997 Sep 26, 204(2), 117 - 23
Modification of methane emission in sheep by cysteine and a microbial preparation; Takahashi J et al.; Modification of methane emission from ruminants by the use of L-cysteine and/or a microbial preparation in wethers fed lucerne hay was assessed in an open circuit respiratory trial according to a 4 x 4 Latin squared design . L-Cysteine (0.1 g kg-1 W0.75, as sulphur equivalent) was drenched daily to wethers as an aqueous solution of hydrochloride . The microbial preparation was a dried mixture of Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Pseudomonas fluorescens, Streptomyces cellulosae, Streptomyces albidoflavus and Saccharomyces lypolyticum . The microbial preparation was added at 87 mg kg-1 metabolic weight (W0.75) to the daily diet of a wether sheep offered lucerne chaffed hay at a maintenance level of 54 g DM kg-1 W0.75 . L-Cysteine suppressed methane emission from the sheep by 13% whereas the microbial preparation actually increased methane emission by 18% . Oxidation-reduction potential, pH, ammonia and VFA were non-significantly influenced by the applied treatments.

Gene, 1997 Aug 11, 195(1), 49 - 53
Cloning of the phosphonoacetate hydrolase gene from Pseudomonas fluorescens 23F encoding a new type of carbon-phosphorus bond cleaving enzyme and its expression in Escherichia coli and Pseudomonas putida; Kulakova AN et al.; The phnA gene encoding a novel carbon-phosphorus bond cleavage enzyme, phosphonoacetate hydrolase, from Pseudomonas fluorescens 23F was cloned and expressed in Escherichia coli and Pseudomonas putida . It conferred on the latter host the ability to mineralize phosphonoacetate but on the former the ability to utilize it as sole phosphorus source only . The nucleotide and deduced amino acid sequences of the phnA gene showed no significant homology with any data bank accessions.

Appl Microbiol Biotechnol, 1997 Aug, 48(2), 177 - 83
Family-10 and family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps; Clarke JH et al.; Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated . The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases . Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness . None of the enzymes had a deleterious effect on pulp fibre integrity . The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan . Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated . Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases . Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp . cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp.

FEMS Microbiol Lett, 1997 Aug 15, 153(2), 363 - 9
High-efficiency transposon mutagenesis by electroporation of a Pseudomonas fluorescens strain; Artiguenave F et al.; A method is described for mutagenesis of Pseudomonas fluorescens strains by electroporation with the transposon delivery vector pUT/mini-Tn5 Km . The transposition process was shown to be optimal at 12.5 kV cm-1 for a pulse time (Bowen and Koslak, 1992) of about 4 ms . The Pseudomonas fluorescens L6.5 target strain exhibited maximal electrocompetence when harvested at the middle of the exponential growth phase . As many as 7.7 10(5) mutants per picomole of delivery vector (7.5 kb) could be obtained, and these kanamycin-resistant mutants were shown to have lost the pUT plasmid . By external calibration with plasmids of increasing size (from 11.5 to 60.1 kb), the efficiency of the transformation process was evaluated to be approximately 1.31 x 10(8) transformants per picomole of delivery vector . Efficiency of the transposition process was 0.58% . This rapid method was used to tag for the cloning three independent chromosomal loci responsible for the Alk+ phenotype of Pseudomonas fluorescens L6.5 strain.

FEMS Microbiol Lett, 1997 Aug 15, 153(2), 357 - 61
Fluviols, bicyclic nitrogen-rich antibiotics produced by Pseudomonas fluorescens; Smirnov VV et al.; Fifteen fluorescent pseudomonads, isolated from the rhizosphere of agricultural plants, were similar in both their phenotypic properties and the chemical nature of produced pigments, to the previously described Pseudomonas fluorescens var . pseudoiodinum . DNA-DNA hybridisation data showed their genetic similarity (but not identity) to different biovars of P . fluorescens . A family of antibiotics-fluviols belonging to pyrazolo-{4,3-e}as-triazine derivatives was isolated from studied strains; isolation, properties, antimicrobial and antitumour activity of fluviols are described.

Arch Biochem Biophys, 1997 Aug 15, 344(2), 301 - 8
Cloning, sequence, and expression of kynureninase from Pseudomonas fluorescens; Koushik SV et al.; We have cloned the gene encoding kynureninase from Pseudomonas fluorescens using a restriction site polymerase chain reaction technique (RS-PCR) (G . Sarkar, R . T . Turner, and M . E . Bolander PCR Methods Appl . 2, 318-322, 1993) and expressed the enzyme in Escherichia coli DH5a F' . The kynureninase gene has an open reading frame (ORF) of 1251 base pairs that codes for a protein of 416 amino acids with a calculated molecular weight of 45,906 . The protein purified from P . fluorescens has N-terminal threonine and an observed molecular weight of 45,787 by electrospray mass spectrometry, suggesting that the N-terminal methionine is removed by posttranslational processing . The complete gene was obtained by PCR and inserted into pTZ18U . The resultant plasmid was used to transform E . coli DH5alpha F', and these cells overexpressed kynureninase to about 37% of total soluble protein . The isolated recombinant protein has molecular weight and Km values identical to those of the native protein from P . fluorescens . The amino acid sequence exhibits 29% identity with those of rat and human kynureninases and 32% identity with the amino acid sequence translated from a Saccharomyces cerevisiae ORF . Alignment of the four sequences shows a highly conserved region which corresponds to the pyridoxal-5'-phosphate (PLP) binding site of rat kynureninase . Based on this alignment, we predict that Lys227 and Asp212 in P . fluorescens kynureninase are involved in pyridoxal-5'-phosphate binding . P . fluorescens kynureninase also exhibits significant homology to the nifS gene product, cysteine desulfurase, and to eucaryotic serine/pyruvate aminotransferases, suggesting that it is a member of subgroup IV of the aminotransferase family of PLP-dependent enzymes.

Biochem J, 1997 Aug 1, 325 ( Pt 3), 761 - 9
Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA; Garcia I et al.; p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate . In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones . Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides . In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells . Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol . Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45-46 kDa . This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens . Gel-filtration chromatography indicates that the enzyme behaves as a homodimer . We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase . The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases . This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells . Comparison of the N-terminal sequence of the 45-46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity . Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification . This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

Curr Microbiol, 1997 Aug, 35(2), 97 - 102
Detection and molecular analysis of plant- and insect-associated bacteria harboring aconitate isomerase involved in biosynthesis of trans-aconitic acid as antifeedant in brown planthoppers; Watanabe K et al.; The activity of aconitate isomerase, which is involved in the biosynthesis of trans-aconitic acid as antifeedant in brown planthoppers, was detected in Pseudomonas fluorescens LRB3W1 and Pseudomonas putida MAFF301685 but not in Pseudomonas putida MAFF301684 . The enzyme activity was induced in the presence of trans-aconitate, and therefore bacteria showing the enzyme activity were easily detected by their ability to grow on the minimal medium containing trans-aconitate as the sole carbon source (ACO agar medium) . Experiments on growth of plant- or insect-associated bacteria on ACO agar medium showed that most of the Gram-negative bacteria displayed the aconitate isomerase activity unlike most of the Gram-positive bacteria isolated mainly from insects . Mini-Tn5 transposon derivatives of P . fluorescens LRB3W1 lacking completely or partially their ability to grow on ACO agar medium were obtained . Southern blot analysis with a mini-Tn5 DNA probe definitely showed that the genes responsible for the biosynthesis of aconitate isomerase present on chromosomal DNA . Thus, it was suggested that genes for aconitate isomerase biosynthesis are commonly present in Gram-negative plant- or insect-associated bacteria, and also the DNA fragments including the genes were detected in P . fluorescens LRB3W1.

Mol Plant Microbe Interact, 1997 Jul, 10(5), 580 - 8
Evidence that the Pseudomonas syringae pv . syringae hrp-linked hrmA gene encodes an Avr-like protein that acts in an hrp-dependent manner within tobacco cells; Alfano JR et al.; A 25-kb DNA region, previously cloned from Pseudomonas syringae pv . syringae 61 in cosmid pHIR11, enables nonpathogenic bacteria such as Pseudomonas fluorescens and Escherichia coli to elicit the hypersensitive response (HR) in tobacco (Nicotiana tabacum) . hrmA is located within this region, adjacent to a conserved cluster of hrp genes, and is essential for nonpathogens to elicit the HR . DNA sequence analysis suggested that hrmA was the second of two genes in an operon and was preceded by an open reading frame (ORF), ORF1, which is predicted to encode a 10.9-kDa protein . DNA gel blot analysis revealed that sequences hybridizing with a DNA fragment internal to hrmA were absent from P . syringae pv . syringae B728a, P . syringae pv . tabaci 11528, and P . syringae pv . glycinea race 4 U1, but present in P . syringae pv . tomato DC3000 . A 2.4-kb BamHI-AvrII fragment carrying hrmA, ORF1, and native regulatory sequences was subcloned into broad-host-range vector pDSK519 and electroporated into P . syringae pv . syringae B728a and P . syringae pv . tabaci 11528 . The presence of the hrmA locus had no apparent effect on the ability of P . syringae pv . syringae B728a to cause brown spot of bean, but it caused P . syringae pv . tabaci 11528 to elicit the defense-associated HR rather than disease in N . tabacum cvs . Xanthi N and Xanthi NC and N . clevelandii . Furthermore, N . debeyii, N . glutinosa, N . rustica, and N . tabacum cvs . Petit Havana and Samsun responded with the HR to P . fluorescens(pHIR11) . In contrast, N . benthamiana-P . syringae pv . tabaci interactions were unaffected by the presence of HrmA, and P . fluorescens(pHIR11) did not elicit the HR in N . benthamiana . The hrmA ORF was subcloned into pFLAG-CTC, which expressed HrmA with a C-terminal FLAG synthetic epitope fusion . Escherichia coli MC4100 cells carrying the functional hrp cluster and the hrmA-FLAG derivative secreted the HrpZ harpin, but not HrmA-FLAG, to the medium, as indicated by immunoblot analysis with M2 anti-FLAG and polyclonal anti-HrpZ antibodies . The hrmA ORF was also subcloned into plant expression vector pFF19 and then biolistically delivered, along with pFF19G (expressing beta-glucuronidase), into suspension-cultured tobacco cells . Histochemical staining 24 h later revealed substantial beta-glucuronidase activity in cells receiving pFF19G and pFF19 but not in those receiving pFF19G and pFF19-HrmA . Thus, internal production of HrmA was deleterious to tobacco cells.

MMWR Morb Mortal Wkly Rep, 1997 Jun 20, 46(24), 553 - 5
Red blood cell transfusions contaminated with Yersinia enterocolitica--United States, 1991-1996, and initiation of a national study to detect bacteria-associated transfusion reactions; Factors contributing to the survival of poultry associated Pseudomonas spp . exposed to a quaternary ammonium compound; MATFORSK, Norwegian Food Research Institute, As, NorwayResistance to benzalkonium chloride (BC) among Pseudomonas spp . isolated from poultry carcasses was determined and strategies for elimination of resistant strains evaluated . This investigation showed that resistance was quite common, about 30% of the isolates being able to grow in 200 micrograms ml-1 BC . Pseudomonas fluorescens strains were generally less susceptible than strains of Ps . lundensis and Ps . fragi . An overnight incubation in medium containing 200 micrograms ml-1 BC was sufficient to reduce the susceptibility of two Pseudomonas strains to the lethal effect of BC significantly . Adding EDTA enhanced the lethal effect of BC, but the effect was reduced after growing cells in medium containing BC and EDTA . Growth in medium with a quaternary ammonium compound (QAC) rendered the cells more susceptible to chlorine, phenolics, and alkylaminoacetate . These results indicate that alternating use of QACs with these compounds can be used to avoid build-up of resistant strains . In addition, increased temperatures improved the lethal effect of BC and should be considered when planning disinfection routines.

Appl Environ Microbiol, 1997 Jun, 63(6), 2232 - 9
Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST; Beltrametti F et al.; The nucleotide sequence of the 4,377-bp chromosomal region of Pseudomonas fluorescens ST that codes for the oxidation of styrene to phenylacetic acid was determined . Four open reading frames, named styA, styB, styC, and styD, were identified in this region . Sequence analysis and biotransformation assays, performed with batch and continuous cultures, allowed us to identify the functions of the sequenced genes . styA and styB encode a styrene monooxygenase responsible for the transformation of styrene to epoxystyrene; styC codes for the second enzyme of the pathway, an epoxystyrene isomerase that converts epoxystyrene to phenylacetaldehyde; and the styD gene produces a phenylacetaldehyde dehydrogenase that oxidizes phenylacetaldehyde to phenylacetic acid . StyA, 415-amino-acids long, was found to be weakly homologous to p-hydroxybenzoate hydroxylase from both P . fluorescens and P . aeruginosa and to salicylate hydroxylase from P . putida, suggesting that it might be a flavin adenine dinucleotide-binding monooxygenase . StyB was found to be partially homologous to the carboxyterminal part of the 2,4-dichlorophenol-6-monooxygenase encoded by plasmid pJP4, while the styC product did not share significant homology with any known proteins . The fourth open reading frame, styD, could encode a protein of 502 amino acids and was strongly homologous to several eukaryotic and prokaryotic aldehyde dehydrogenases . The order of the genes corresponds to that of the catabolic steps . The previously suggested presence of the gene for epoxystyrene reductase, which directly converts epoxystyrene to 2-phenylethanol (A.M . Marconi, F . Beltrametti, G . Bestetti, F . Solinas, M . Ruzzi, E . Galli, and E . Zennaro, Appl . Environ . Microbiol . 61:121-127, 1996), has not been confirmed by sequencing and by biotransformation assays performed in continuous cultures . A copy of the insertion sequence ISI162, belonging to the IS21-like family of elements, was identified immediately downstream of the styrene catabolic genes.

Carbohydr Res, 1997 May 19, 300(4), 323 - 7
The structure of the exopolysaccharide of Pseudomonas fluorescens strain H13; Osman SF et al.; An acidic exopolysaccharide was isolated from P . fluorescens strain H13 . The structure of the polysaccharide repeating unit was determined using chemical methods and 1D and 2D NMR techniques . The repeating unit was characterized as a trisaccharide composed of D-glucose, 2-acetamido-2-deoxy-D-glucose and 4-O-acetyl-2-acetamido-2-deoxy-D-mannuronic acid.

J Biotechnol, 1997 May 9, 54(3), 151 - 60
Investigation of refolding condition for Pseudomonas fluorescens lipase by response surface methodology; Ahn JH et al.; Response Surface Methodology (RSM) was used to investigate optimal refolding conditions of Pseudomonas fluorescens lipase which was expressed as inclusion body in E . coli . Three interacting factors, protein concentration, pH and guanidine hydrochloride (GdnHCl) concentration were selected as the variables of RSM . The protein concentration did not affect the refolding yield within the selected range (50-340 micrograms ml-1) at low temperatures of 4 and -15 degrees C, but it was a critical factor at 25 degrees C and refolding yield significantly decreased with increasing protein concentration . The pH and GdnHCl were significant factors in all experimental conditions . But there was no trends of optimal pH depending on temperature and additives, just showing the optimum at neutral pH . Glycerol shifted optimal GdnHCl concentration to higher side, while arginine to lower side . Therefore, it was concluded that glycerol and arginine deprives and supplements GdnHCl, respectively . In this study, RSM made it possible to investigate successfully the optimal conditions of in vitro refolding and to elucidate interactions between refolding factors with a minimum number of experiments . Under the optimal condition determined by RSM, approximately 90% refolding yield was obtained and it was a 30% increase over the conventional method.

Lett Appl Microbiol, 1997 May, 24(5), 329 - 33
Total biodegradation of the oestrogenic mycotoxin zearalenone by a bacterial culture; Megharaj M et al.; A mixed culture of bacteria, enriched from soil collected at a coal gasification site, proved capable of removing the potent oestrogenic mycotoxin zearalenone from culture media . The bacteria grew rapidly when zearalenone was provided as the sole source of carbon and energy . HPLC and ELISA analysis of culture extracts revealed no zearalenone or zearalenone-like products . Fourteen bacterial isolates from the mixed culture were identified and purified . The ability to degrade zearalenone was lost upon purification and recombination of the bacterial members of the mixed culture . A strain of Pseudomonas fluorescens capable of degrading polychlorinated biphenyls was unable to degrade zearalenone . This is the first report of the complete degradation of zearalenone by bacteria . The present study suggests the potential of mixed cultures in the biodegradation of zearalenone.

J Bacteriol, 1997 May, 179(9), 3036 - 8
In vivo construction of a hybrid pathway for metabolism of 4-nitrotoluene in Pseudomonas fluorescens; Michan C et al.; Pseudomonas fluorescens 410PR grows on 4-nitrobenzoate but does not metabolize 4-nitrotoluene . The TOL pWW0 delta pm plasmid converts 4-nitrotoluene into 4-nitrobenzoate through its upper pathway, but it does not metabolize 4-nitrobenzoate . P . fluorescens 410PR(pWW0 delta pm) transconjugants were isolated and found to be able to grow on 4-nitrotoluene . This phenotype was stable after growth for at least 300 generations without any selective pressure . P . fluorescens 410PR(pWW0 delta pm) converted 4-nitrotoluene into 4-nitrobenzoate via 4-nitrobenzylalcohol and 4-nitrobenzaldehyde . 4-Nitrobenzoate was metabolized via 4-hydroxylaminobenzoate and finally yielded NH4+ and 3,4-dihydroxybenzoate, which was mineralized.

Biochem J, 1997 Apr 15, 323 ( Pt 2), 547 - 55
Arabinanase A from Pseudomonas fluorescens subsp . cellulosa exhibits both an endo- and an exo- mode of action; McKie VA et al.; Pseudomonas fluorescens subsp . cellulosa expressed arabinanase activity when grown on media supplemented with arabinan or arabinose . Arabinanase activity was not induced by the inclusion of other plant structural polysaccharides, and was repressed by the addition of glucose . The majority of the Pseudomonas arabinanase activity was extracellular . Screening of a genomic library of P . fluorescens subsp . cellulosa DNA constructed in Lambda ZAPII, for recombinants that hydrolysed Red-dyed arabinan, identified five arabinan-degrading plaques . Each of the phage contained the same Pseudomonas arabinanase gene, designated arbA, which was present as a single copy in the Pseudomonas genome . The nucleotide sequence of arbA revealed an open reading frame of 1041 bp encoding a protein, designated arabinanase A (ArbA), of Mr 39438 . The N-terminal sequence of ArbA exhibited features typical of a prokaryotic signal peptide . Analysis of the primary structure of ArbA indicated that, unlike most Pseudomonas plant cell wall hydrolases, it did not contain linker sequences or have a modular structure, but consisted of a single catalytic domain . Sequence comparison between the Pseudomonas arabinanase and proteins in the SWISS-PROT database showed that ArbA exhibits greatest sequence identity with arabinanase A from Aspergillus niger, placing the enzyme in glycosyl hydrolase Family 43 . The significance of the differing substrate specificities of enzymes in Family 43 is discussed . ArbA purifed from a recombinant strain of Escherichia coli had an Mr of 34000 and an N-terminal sequence identical to residues 32-51 of the deduced sequence of ArbA, and hydrolysed linear arabinan, carboxymethylarabinan and arabino-oligosaccharides . The enzyme displayed no activity against other plant structural polysaccharides, including branched sugar beet arabinan . ArbA produced almost exclusively arabinotriose from linear arabinan and appeared to hydrolyse arabino-oligosaccharides by successively releasing arabinotriose . ArbA and the Aspergillus arabinanase mediated a decrease in the viscosity of linear arabinan that was associated with a significant release of reducing sugar . We propose that ArbA is an arabinanase that exhibits both an endo- and an exo- mode of action.

Arch Biochem Biophys, 1997 Apr 15, 340(2), 168 - 76
Catalytic properties of lipoamide dehydrogenase from Mycobacterium smegmatis; Marcinkeviciene J et al.; Lipoamide dehydrogenase from Mycobacterium smegmatis was purified to homogeneity over 60-fold . Of 20 amino acid residues identified at the amino terminus of the enzyme, 18 and 17 were identical to the sequences of Mycobacterium leprae and Pseudomonas fluorescens lipoamide dehydrogenases, respectively . The visible spectrum of the isolated enzyme was characteristic of a flavin in apolar environment . Reduction of the enzyme with dithionite results in the appearance of an absorbance shoulder at 530-550 nm, suggesting that reducing equivalents of the two-electron reduced enzyme reside predominantly on the redox-active disulfidedithiol . The kinetic mechanism of the forward (NAD+ reducing) and reverse (NADH oxidizing) reactions proved difficult to study due to severe substrate inhibition by NAD+ and NADH . The rate of lipoamide reduction was found to depend upon the NAD+/NADH ratio, with the reaction being activated at low ratios and inhibited at high ratios . The use of 3-acetylpyridine adenine dinucleotide allowed initial velocity kinetics to be performed and revealed that the kinetic mechanism is ping pong . In addition to catalyzing the reversible oxidation of dihydrolipoamide, the enzyme displayed high oxidase activity (30% of the lipoamide reduction rate), hydrogen and t-butyl peroxide reductase activity (10% of the lipoamide reduction rate), and both naphthoquinone and benzoquinone reduction (approximately 200% of the lipoamide reduction rate) . The enzyme failed to catalyze the redox cycling of nitrocompounds, but could anaerobically reduce nitrofurazone . The lipoamide-reducing reaction was reversibly inactivated by sodium arsenite, but no decrease in diaphorase activity was observed under these conditions.

Biochemistry, 1997 Apr 8, 36(14), 4163 - 71
Structures and characteristics of novel siderophores from plant deleterious Pseudomonas fluorescens A225 and Pseudomonas putida ATCC 39167; Khalil-Rizvi S et al.; When Pseudomonas putida ATCC 39167 and plant-deleterious Pseudomonas fluorescens A225 were grown in an iron-deficient culture medium, they each produced two different novel yellow-green fluorescent pseudobactins: P39167-I, II and PA225-I, II . Pseudobactin P39167-I has a molecular formula of C46H65O23N13 and is monoanionic at neutral pH . P39167-II has the molecular formula of C46H63O22N13 and no charge at neutral pH . Pseudobactin PA225-I has a molecular formula of C46H65O24N13 and is monoanionic at neutral pH whereas pseudobactin PA225-II has the molecular formula of C46H63O23N13 and no charge at neutral pH . All four of the pseudobactins contain a dihydroxyquinoline-based chromophore . The amino acid sequence for the octapeptide in case of pseudobactins from P . putida ATCC 39167 is Chr-Ser(1)-Ala(1)-AcOHOrn-Gly-Ala(2)-OHAsp-Ser(2)-Thr . In case of pseudobactins from P . fluorescens A225, the octapeptide has the sequence Chr-Ser(1)-Ala-AcOHOrn-Gly-Ser(2)-OHAsp-Ser(3)-Thr . For all four pseudobactins (P39167-I, II and PA225-I, II), the serine(1) residue of the octapeptide is attached to the carboxylic acid group on the C-11 of the fluorescent quinoline via an amide bond . Additionally, for pseudobactin P39167-II and PA225-II, the hydroxyl group of the serine(1) residue is also attached to the carboxyl group of threonine residue at the carboxy terminus of the peptide via an ester bond, resulting in a cyclic depsipeptide in contrast to the linear peptide chain of P39167-I and PA225-I . For all four pseudobactins, a malamide group is attached to the C-3 of the quinoline derived chromophore . The three bidentate iron(III) chelating groups in all four pseudobactins consist of a 1,2-dihydroxy aromatic group of the fluorescent chromophore, a hydroxy acid group of beta-hydroxy aspartic acid, and a hydroxamate group from the acylated Ndelta-hydroxyornithine . The amino acid constituents of the pseudobactins P39167 I, II are the same as those in pseudobactin A214, whereas those in A225 I, II are the same as in 7SR1, but in both cases the sequences are different . The uptake results indicate a single outer membrane receptor protein for ferric-pseudobactins in both organisms . The receptor proteins in the two species are similar but not identical.

Can J Microbiol, 1997 Apr, 43(4), 368 - 77
Comparative study of five polycyclic aromatic hydrocarbon degrading bacterial strains isolated from contaminated soils; Dagher F et al.; Five polycyclic aromatic hydrocarbon (PAH) degrading bacterial strains, Pseudomonas putida 34, Pseudomonas fluorescens 62, Pseudomonas aeruginosa 57, Sphingomonas sp . strain 107, and the unidentified strain PL1, were isolated from two contaminated soils and characterized for specific features regarding PAH degradation . Degradation efficiency was determined by the rapidity to form clearing zones around colonies when sprayed with different PAH solutions and the growth in liquid medium with different PAHs as sole source of carbon and energy . The presence of plasmids, the production of biosurfactants, the effect of salicylate on PAH degradation, the transformation of indole to indigo indicating the presence of an aromatic ring dioxygenase activity, and the hybridization with the SphAb prove representing a sequence highly homologous to the naphthalene dioxygenase ferredoxin gene nahAb were examined . The most efficient strain in terms of substrate specificity and rapidity to degrade different PAHs was Sphingomonas sp . strain 107, followed by strain PL1 and P . aeruginosa 57 . The less efficient strains were P . putida 34 and P . fluorescens 62 . Each strain transformed indole to indigo, except strain PL1 . Biosurfactants were produced by P . aeruginosa 57 and P . putida 34, and a bioemulsifier was produced by Sphingomonas sp . strain 107 . The presence of salicylate in solid medium has accelerated the formation of clearing zones and the transformation of indole by Sphingomonas sp . strain 107 and P . aeruginosa 57 colonies . Plasmids were found in Sphingomonas sp . strain 107 and strain PL1 . The SphAb probe hybridized with DNA extracted from each strain . However, hybridization signals were detected only in the plasmidic fraction of Sphingomonas sp . strain 107 and strain PL1 . Using a polymerase chain reaction (PCR) approach, we determined that several genes encoding enzymes involved in the upper catabolic pathway of naphthalene were present in each strain . Sequencing of PCR DNA fragments revealed that, for all the five strains, these genes are highly homologous with respective genes found in the pah, dox, and nah operons, and are arranged in a polycistronic operon . Results suggest that these genes are ordered in the five selected strains like the pah, nah, and dox operons.

Can J Microbiol, 1997 Apr, 43(4), 344 - 53
Bacterial communities of the rhizosphere and endorhiza associated with field-grown cucumber plants inoculated with a plant growth-promoting rhizobacterium or its genetically modified derivative; Mahaffee WF et al.; The future use of genetically modified microorganisms in the environment will be dependent on the ability to asses potential or theoretical risks associated with their introduction into natural ecosystems . To assess potential risks, several ecological parameters must be examined, including the impact of the introduced genetically modified organism on the microbial communities associated with the environment into which the introduction will occur . A 2-year field study was established to examine whether the indigenous bacterial communities of the rhizosphere and endorhiza (internal root tissues) were affected differently by the introduction of an unaltered wild type and its genetically modified derivative . Treatments consisted of the wild-type strain Pseudomonas fluorescens 89B-27 and a bioluminescent derivative GEM-8 (89B-27::Tn4431) . Cucumber root or seed samples were taken 0, 7, 14, 21, 35, and 70 days after planting (DAP) in 1994 and 0, 7, 14, 28, 42, and 70 DAP in 1995 . Samples were processed to examine the bacterial communities of both the rhizosphere ad endorhiza . Over 7200 bacterial colonies were isolated from the rhizosphere Community structure at the genus level was assessed using genera richness and Hill's diversity numbers, N1 and N2 . The aerobic-heterotrophic bacterial community structure at the genus level did not significantly vary between treatments but did differ temporally . The data indicate that the introduction of the genetically modified derivative of 89B-27 did not pose a greater environmental risk than its unaltered wild type with respect to aerobic-heterotrophic bacterial community structure.

Biochem Mol Biol Int, 1997 Apr, 41(4), 821 - 31
Molecular cloning and analysis of a HSP (heat shock protein)-like 42 kDa antigen gene of Mycoplasma hyopneumoniae; Chou SY et al.; The recombinant clone expressing the 42 kDa protein (P42) of Mycoplasma hyopneumoniae in Escherichia coli was analyzed . The 4.4 kb HindIII-Xmal DNA fragment expressing the p42 gene product encodes three ORFs: p42 and p16 in the forwarding strand, p24 in the reverse strand . Sequence comparisons revealed that p42 could be part of a p65 gene, and has 62% identities with Mycoplasma genitalium HSP70 gene and 56% identities with Bacillus subtilis dnaK gene; p16 and p24 genes share 73% and 47% identities with Erysipelothrix rhusiopathiae dnaJ gene and Pseudomonas fluorescens uvrC gene, respectively . Further analysis demonstrated that P42 is indeed a heat shock protein and the monospecific antibodies against P42 can block the growth of Mycoplasma hyopneumoniae.

J Bacteriol, 1997 Apr, 179(8), 2761 - 5
Cloning, sequence, and properties of the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens; French CE et al.; The gene encoding the soluble pyridine nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli . STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues . The gene is highly similar to an E . coli gene of unknown function.

Appl Environ Microbiol, 1997 Apr, 63(4), 1617 - 22
Comparative analysis of antibiotic resistance, immunofluorescent colony staining, and a transgenic marker (bioluminescence) for monitoring the environmental fate of rhizobacterium; Mahaffee WF et al.; Field releases of the wild-type plant growth-promoting rhizobacterium Pseudomonas fluorescens 89B-27, its bioluminescent derivative GEM-8 (89B-27::Tn4431), and a spontaneous rifampin-resistant variant estimating the wild-type population . Seed and root samples were taken 0, 7, 14, 21, or 28, 35 or 42, and 70 days after planting in each year and processed for enumeration by spiral plating or immunofluorescent colony staining (IFC) . In both years, the populations of 89B-27, R34, and GEM-8, as measured by IFC, were not significantly different (P > 0.05) from each other at each sampling time . However, the populations of R34 and GEM-8, as measured by spiral plating and differentiation based on their respective phenotypes, were significantly lower (P < 0.05) than the wild-type populations and their IFC-determined populations . These data indicate that traditional marker systems may underestimate populations and hence the survival and colonization of genetically marked bacteria.

Appl Environ Microbiol, 1997 Apr, 63(4), 1332 - 7
Plasmid-mediated mineralization of carbofuran by Sphingomonas sp . strain CF06; Feng X et al.; A bacterial strain (CF06) that mineralized both the carbonyl group and the aromatic ring of the insecticide carbofuran and that is capable of using carbofuran as a sole source of carbon and nitrogen was isolated from a soil in Washington state . Phospholipid fatty acid and 16S rRNA sequencing analysis indicate that CF06 is a Sphingomonas sp . CF06 contains five plasmids, at least some of which are required for metabolism of carbofuran . Loss of the plasmids induced by growth at 42 degrees C resulted in the inability of the cured strain to grow on carbofuran as a sole source of carbon . Introduction of the plasmids confers on Pseudomonas fluorescens M480R the ability to use carbofuran as a sole source of carbon for growth and energy . Of the five plasmids, four are rich in insertion sequence elements and contain large regions of overlap . Rearrangements, deletions, and loss of individual plasmids that resulted in the loss of the carbofuran-degrading phenotype were observed following introduction of Tn5.

Microbiology, 1997 Mar, 143 ( Pt 3), 1029 - 35
Growth temperature dependence of channel size of the major outer-membrane protein (OprF) in psychrotrophic Pseudomonas fluorescens strains; De E et al.; The outer-membrane (OM) permeability of the psychrotrophic bacterium Pseudomonas fluorescens strain MF0 for the beta-lactam mezlocillin is increased at the optimum growth temperature (28 degrees C) compared to low growth temperatures (8 degrees C) . In an attempt to explain this phenomenon, OM protein content was studied in cultures grown at both temperatures . No significant difference in proportion or composition was found, suggesting that a change in the structure and function of porins could be responsible for the differential permeability . The major OM protein OprF of two psychrotrophic P . fluorescens strains, MF0 and OE 28.3, was purified from cultures grown at 8 degrees C and 28 degrees C in order to reincorporate them in solvent-free lipid bilayers . From cultures grown at the same temperature, OprF displayed very similar channel-forming properties for both strains . Decreasing the growth temperature induced a threefold reduction of the major conductance values (250-270 pS in 1 M NaCl for 28 degrees C cultures and 80-90 pS in 1 M NaCl for 8 degrees C cultures) . The trypsin digestion kinetics showed a very different reactivity for these porins between cultures grown at 8 degrees C and 28 degrees C . This may indicate that the pore structure of OprF is modified depending on the growth temperature, as suggested by its functional behaviour.

Microbiology, 1997 Mar, 143 ( Pt 3), 1019 - 27
A phosphate-starvation-inducible outer-membrane protein of Pseudomonas fluorescens Ag1 as an immunological phosphate-starvation marker; Leopold K et al.; A phosphate-starvation-inducible outer-membrane protein of Pseudomonas fluorescens Ag1, expressed at phosphate concentrations below 0.08-0.13 mM, was purified and characterized . The purification method involved separation of outer-membrane proteins by SDS-PAGE and extraction of the protein from nitrocellulose or PVDF membranes after electrotransfer of proteins to the membranes . The N-terminal amino acid sequence of the purified protein, called Psi1, did not show homology to any known proteins, and in contrast to the phosphate-specific porin OprP of P . aeruginosa its mobility in SDS-PAGE was not affected by solubilization temperature . An antiserum against Psi1 recognized a protein of M, 55,000 in four other P . fluorescens strains among 24 tested strains representing Pseudomonas rRNA homology group I, showing antigenic heterogeneity within this group . A method for immunofluorescence microscopy involving cell permeabilization was adapted to visualize cell-specific expression of Psi1 in P . fluorescens exposed to limiting amounts of phosphate . This approach should be useful for further exploration of Psi1 as a marker to study the availability of phosphate to P . fluorescens in natural environments.

Lett Appl Microbiol, 1997 Mar, 24(3), 198 - 202
The flagellin gene as a stable marker for detection of Pseudomonas fluorescens SBW25; Denning N et al.; Flagellin gene central regions from 111 isolates of Pseudomonas fluorescens SBW25 obtained from soil during a field release experiment were analysed using a combined PCR/RFLP technique to look for variation . In addition, a 858 bp flagellin gene sequence from the original strain and the last isolate obtained from the release site were compared . There was no variation in flagellin gene sequences indicating that the gene was stable over the period of the release, and that the flagellin gene is a suitable marker for use in the detection of bacteria in release experiments . A comparison of Ps . fluorescens SBW25 flagellin with other sequenced flagellins revealed closest homology to the flagellin of Ps . putida PRS2000.

Gene, 1997 Feb 28, 186(2), 167 - 73
Chromosomal insertion of the entire Escherichia coli lactose operon, into two strains of Pseudomonas, using a modified mini-Tn5 delivery system; Hansen LH et al.; A 12-kb PstI fragment including the entire E . coli lactose operon (lacIPOZYA) was inserted in one copy into the chromosome of Pseudomonas putida, Pseudomonas fluorescens and an E . coli strain with lac- phenotype . This was made possible by improvements of an already existing mini-Tn5 transposon delivery system (de Lorenzo et al., 1990; Herrero et al., 1990), which integrates cloned DNA fragments at random sites on the chromosome of the recipient bacteria in single copies . This has resulted in: (a) the making of two useful low copy-number cloning vectors both with extensive multi-cloning regions flanked by NotI sites needed in the mini-Tn5 delivery system; (b) the generation of E . coli nonlysogenic strains expressing the pi protein thus being capable of maintaining and delivering R6K-based mini-Tn5 vectors to other E . coli strains; (c) the successful insertion of the E . coli lactose operon into the P . fluorescens chromosome giving P . fluorescens the ability to grow on lactose; (d) evidence from Southern blotting that contradicts the assumption that the mini-Tn5 delivery system always creates one-copy inserts . These improvements allow insertion of large DNA fragments encoding highly expressed proteins into the chromosome of a large variety of Gram-negative bacteria including E . coli.

Biochim Biophys Acta, 1997 Feb 28, 1350(3), 272 - 6
Purification of the dissimilative nitrate reductase of Pseudomonas fluorescens and the cloning and sequencing of its corresponding genes; Philippot L et al.; The dissimilative membrane-bound nitrate reductase from Pseudomonas fluorescens strain AK15 was purified and the alpha subunit of the enzyme partially sequenced . On the basis of this partial amino acid sequence and of conserved stretches of amino acids between Escherichia coli and Bacillus subtilis, degenerate primers were design to amplify the narG gene and part of the narH gene in a PCR approach . The deduced amino acid sequence of narG shows 72% and 52% and narH 78% and 62% identity to the homologous subunit of E . coli and B . subtilis, respectively.

J Biol Chem, 1997 Jan 31, 272(5), 2942 - 51
Key residues in subsite F play a critical role in the activity of Pseudomonas fluorescens subspecies cellulosa xylanase A against xylooligosaccharides but not against highly polymeric substrates such as xylan; Charnock SJ et al.; In a previous study crystals of Pseudomonas fluorescens subspecies cellulosa xylanase A (XYLA) containing xylopentaose revealed that the terminal nonreducing end glycosidic bond of the oligosaccharide was adjacent to the catalytic residues of the enzyme, suggesting that the xylanase may have an exo-mode of action . However, a cluster of conserved residues in the substrate binding cleft indicated the presence of an additional subsite, designated subsite F . Analysis of the biochemical properties of XYLA revealed that the enzyme was a typical endo-beta1,4-xylanase, providing support for the existence of subsite F . The three-dimensional structure of four family 10 xylanases, including XYLA, revealed several highly conserved residues that are on the surface of the active site cleft . To investigate the role of some of these residues, appropriate mutations of XYLA were constructed, and the biochemical properties of the mutated enzymes were evaluated . N182A hydrolyzed xylotetraose to approximately equal molar quantities of xylotriose, xylobiose, and xylose, while native XYLA cleaved the substrate to primarily xylobiose . These data suggest that N182 is located at the C site of the enzyme . N126A and K47A were less active against xylan and aryl-beta-glycosides than native XYLA . The potential roles of Asn-126 and Lys-47 in the function of the catalytic residues are discussed . E43A and N44A, which are located in the F subsite of XYLA, retained full activity against xylan but were significantly less active than the native enzyme against oligosaccharides smaller than xyloseptaose . These data suggest that the primary role of the F subsite of XYLA is to prevent small oligosaccharides from forming nonproductive enzyme-substrate complexes.

J Biochem (Tokyo), 1997 Jan, 121(1), 82 - 8
Purification and characterization of an extracellular metalloprotease from Pseudomonas fluorescens; Kim HJ et al.; An extracellular metalloprotease was purified from the culture supernatant of Pseudomonas fluorescens strain KT1 to apparent homogeneity and shown to consist of a single polypeptide chain (M(r) 46,000-47,000) . The enzyme was strongly inhibited by chelating agents such as EDTA and o-phenanthroline, and activated by certain detergents . Among the peptidyl 4-methylcoumaryl-7-amide (MCA) substrates examined, t-butyloxycarbonyl-Arg-Val-Arg-Arg-MCA was the best one . With this substrate, the enzyme exhibited a pH optimum of around pH 5.5 in the absence of Co2+ ions, whereas it showed two different pH optima (at pHs around 5.5 and 8-9) in the presence of Co2+ ions due to remarkable activation by Co2+ ions in the alkaline pH range . On the other hand, a single broad pH optimum of around 6 to 8 was obtained with some peptides in both the presence and absence of Co2+ ions, and no activation by Co2+ was observed . The enzyme showed trypsin-like specificity, preferentially cleaving certain arginyl peptide bonds, and hydrolyzed the basic protein, histone, most rapidly among various proteins examined . Partial amino acid sequence analysis revealed that the enzyme is highly homologous with proteases of the serralysin family, a group of zinc metalloproteases.

Lett Appl Microbiol, 1997 Jan, 24(1), 5 - 8
Monoclonal antibody detection of Pseudomonas spp . in refrigerated meat by an indirect ELISA; 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 . in refrigerated meat . The detection threshold for the ELISA assay developed in this work was 10(4) cfu cm-2.

Curr Genet, 1997 Jan, 31(1), 22 - 9
Arabinoxylan degradation by fungi: characterization of the arabinoxylan-arabinofuranohydrolase encoding genes from Aspergillus niger and Aspergillus tubingensis; Gielkens MM et al.; The genes encoding the enzyme arabinoxylan arabinofuranohydrolase, which releases L-arabinose from arabinoxylan, have been cloned from the closely related fungi Aspergillus niger and Aspergillus tubingensis and were shown to be functional in A . niger . Integration of multiple copies in the genome resulted in over-expression of the enzymes . The arabinofuranohydrolases encoded comprise 332 amino acids and have 94% amino acid identity . Their primary structure is not related to those of other alpha-L-arabinofuranosidases, except for a low similarity with XYLC, a bacterial alpha-L-arabinofuranosidase from Pseudomonas fluorescens which acts on oat spelt xylan . The axhA expression pattern in A . niger differed from that of abfB, since it was strongly induced by birchwood xylan and much less by L-arabitol or L-arabinose . Furthermore, Northern analysis revealed that axhA expression was de repressed in creAd mutants and carbon catabolite repressed by D-glucose.

Appl Environ Microbiol, 1997 Jan, 63(1), 208 - 12
An Aspergillus niger esterase (ferulic acid esterase III) and a recombinant Pseudomonas fluorescens subsp . cellulosa esterase (Xy1D) release a 5-5' ferulic dehydrodimer (diferulic acid) from barley and wheat cell walls; Bartolome B et al.; Diferulate esters strengthen and cross-link primary plant cell walls and help to defend the plant from invading microbes . Phenolics also limit the degradation of plant cell walls by saprophytic microbes and by anaerobic microorganisms in the rumen . We show that incubation of wheat and barley cell walls with ferulic acid esterase from Aspergillus niger (FAE-III) or Pseudomonas fluorescens (Xy1D), together with either xylanase I from Aspergillus niger, Trichoderma viride xylanase, or xylanase from Pseudomonas fluorescens (XylA), leads to release of the ferulate dimer 5-5' diFA {(E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid} . Direct saponification of the cell walls without enzyme treatment released the following five identifiable ferulate dimers (in order of abundance): (Z)-beta-(4-{(E)-2-carboxyvinyl}-2-methoxyphenoxy)-4-hydroxy-3-methoxycinnamic acid, trans-5-{(E)-2-carboxyvinyl}-2-(4-hydroxy-3-methoxy-phenyl) -7-methoxy-2, 3-dihydrobenzofuran-3-carboxylic acid, 5-5' diFA, (E,E)-4, 4'-dihydroxy-3, 5'-dimethoxy-beta, 3'-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl) -6-methoxy-1, 2-dihydronaphthalene-2, 3-dicarboxylic acid . Incubation of the wheat or barley cell walls with xylanase, followed by saponification of the solubilized fraction, yielded 5-5'diFA and, in some cases, certain of the above dimers, depending on the xylanase used . These experiments demonstrate that FAE-III and XYLD specifically release only esters of 5-5'diFA from either xylanase-treated or insoluble fractions of cell walls, even though other esterified dimers were solubilized by preincubation with xylanase . It is also concluded that the esterified dimer content of the xylanase-solubilized fraction depends on the source of the xylanase.

Appl Environ Microbiol, 1997 Jan, 63(1), 99 - 105
Availability of iron to Pseudomonas fluorescens in rhizosphere and bulk soil evaluated with an ice nucleation reporter gene; Loper JE et al.; The biological availability of iron in the rhizosphere was assessed by evaluating ice nucleation activity (INA) expressed in situ by Pseudomonas fluorescens Pf-5 containing a transcriptional fusion (pvd-inaZ) of an iron-regulated promoter to an ice nucleation reporter gene (inaZ) . Pf-5 containing pvd-inaZ expresses INA that is inversely related to the iron availability of a growth medium (J . E . Loper and S . E . Lindow, Appl . Environ . Microbiol . 60:1934-1941, 1994) . INA expressed by rhizosphere populations of Pf-5 containing pvd-inaZ was at a maximum within 12 to 24 h following inoculation of the bacterium onto bean roots and typically decreased gradually during the following 4 days . Iron availability in the soil, which was altered by the addition of chelators, influenced INA expressed by rhizosphere populations of Pf-5 containing pvd-inaZ . In soil adjusted to a pH of 7.0 or 8.0 by adding Ca(OH)2, rhizosphere populations of Pf-5 containing pvd-inaZ expressed greater INA, indicating lower iron availability, than they did in the nonamended soil at a pH of 5.4 . Similarly, rhizosphere populations of Pf-5 containing pvd-inaZ expressed less INA in an agricultural soil of pH 5.4 than in other agricultural soils ranging in pH from 6.4 to 7.7 . These results conform to the predictions of chemical models stating that pH is a major factor influencing iron availability in soil solutions . The results of this study indicate that P . fluorescens Pf-5 encountered an iron-limited environment immediately after it was inoculated onto bean roots planted in agricultural field soils . One to two days after the bacterium was inoculated onto root surfaces, however, iron became more available to rhizosphere populations of Pf-5 . We speculate that iron acquisition systems of plants and other rhizosphere organisms may provide available sources of iron to established rhizosphere populations of P . fluorescens.

Curr Microbiol, 1997 Jan, 34(1), 12 - 7
A Method for Selection and Characterization of Rhizosphere-Competent Bacteria of Chickpea
Nautiyal CS.
A greenhouse assay was developed to evaluate the root-colonizing capability of the native chickpea rhizospheric bacterial population . In this assay system, screening time was reduced on two counts . First, spontaneous chromosomal rifampicin-resistant (Rifr) strains were directly inoculated to seeds without any check for the stability of the mutation, and second, no attempts were made to taxonomically identify all the strains being screened for chickpea rhizosphere competence . Only two chickpea rhizosphere-competent Rifr strains from the group of six good chickpea rhizosphere colonizers forming 10(7) to 10(8) colony-forming units (cfu)/g root were taxonomically identified as Pseudomonas fluorescens NB13R and Pseudomonas spp . NB49R, after screening 49 bacteria . Both the strains showed no difference from their corresponding wild-type strains P . fluorescens NB13 and Pseudomonas spp . NB49 in terms of chickpea rhizosphere competence . Isogenic or equally rhizospheric competitive second non-isogenic bacterial isolate, when present in tenfold higher amount, pre-empted the colonization of the soil by the bacterium, which was present in smaller ratio . These findings indicate that the isogenic or equally rhizospheric competitive second non-isogenic Rifr strains should be compared for their survival and competition with that of the isogenic parent and with each other for specific ecological niche, before using a mixture of isolates, for stable and consistent biological seed treatment to control soilborn pathogens or pests or to promote plant growth.

Biochemistry, 1996 Dec 17, 35(50), 16195 - 204
Mannanase A from Pseudomonas fluorescens ssp . cellulosa is a retaining glycosyl hydrolase in which E212 and E320 are the putative catalytic residues; Bolam DN et al.; Mannanase A (MANA) from Pseudomonas fluorescens, a member of glycosyl hydrolase family 26, was hyperexpressed in Escherichia coli and purified to homogeneity . Analysis of the stereochemical course of mannotetraose hydrolysis by purified MANA showed that the configuration of the anomeric carbon was retained on cleavage of the middle glycosidic bond . These data suggest that the mannanase hydrolyzes mannooligosaccharides by a double-displacement general acid-base mechanism . By hydrophobic cluster analysis (HCA), two glutamate and two aspartate residues were shown to be conserved in all of the glycosyl hydrolase family 26 enzymes analyzed . In addition, HCA suggested that family 26 was related to the GH-A clan (families 1, 2, 5, 10, 30, 35, 39, and 42) of (alpha/beta)8-barrel glycosyl hydrolases, which led to the prediction that E320 and E212 constitute the catalytic nucleophile and acid-base residues, respectively . To investigate the role of these amino acids, site-directed mutagenesis was used to replace the two aspartates with alanine and glutamate, while the two conserved glutamates were changed to alanine and aspartate . The mutant enzymes were purified and their biochemical properties were analyzed . The data showed that neither the D-->A nor the D-->E mutation resulted in a dramatic decrease in enzyme activity, suggesting that the two aspartate residues did not play a pivotal role in catalysis . In contrast, modification of either of the glutamate residues to alanine caused a dramatic decrease in kcat against carob galactomannan, azo-carob galactomannan, mannotetraose and 2,4-dinitrophenyl beta-mannobioside (2,4-DNPM) . The E320A mutation did not alter the apparent K(m) (K(m)) of MANA against these substrates, while E212A resulted in a 27-fold decrease in K(m) against 2,4-DNPM . Pre-steady-state kinetics of 2,4-DNPM hydrolysis by E212A showed that there was a rapid burst of 2,4-dinitrophenol release . Circular dichroism and fluorescence spectroscopy indicated that there were no significant differences between the structures of the mutant and wild-type forms of MANA . These data are consistent with E212 and E320 constituting the catalytic acid-base and nucleophile residues of MANA, respectively.

J Med Microbiol, 1996 Dec, 45(6), 490 - 3
Indigenous bacterial flora of medicinal leeches and their susceptibilities to 15 antimicrobial agents; Nonomura H et al.; Surface bacterial flora, as well as homogenates, of medical leeches, Hirudo medicinalis and Hirudinaria manillensis, were surveyed and the susceptibility of these isolates to 15 antimicrobial agents was examined . Aeromonas spp . were isolated from all leeches, and Pseudomonas fluorescens and other glucose-non-fermenting gram-negative rods (NF-GNR) were frequent isolates . Isolates were highly resistant to cephalosporins but susceptible to carbapenems, aminoglycosides and ofloxacin . The results indicate that prophylaxis with antimicrobial agents active against Aeromonas spp . and NF-GNR is necessary to avoid opportunist infections caused by indigenous leech flora during medical leech therapy on immunocompromised patients.

Appl Environ Microbiol, 1996 Dec, 62(12), 4471 - 7
Analysis of cumene (isopropylbenzene) degradation genes from Pseudomonas fluorescens IP01; Habe H et al.; We obtained the DNA fragments encoding 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid (HOMODA) hydrolase in the cumene (isopropylbenzene) degrader Pseudomonas fluorescens strain IP01 via PCR using two synthesized oligonucleotides corresponding to the conserved regions within known meta-cleavage compound hydrolases . Following colony hybridization using the amplified DNA as a probe, a 4.5-kb HindIII fragment was isolated from P . fluorescens IP01 . After determining the nucleotide sequence of this fragment, three open reading frames (ORF11 {cumH}, ORF12 {cumD}, and ORF13) were identified . The deduced amino acid sequence of ORF12 showed homology with meta-cleavage compound hydrolases encoded by the tod, dmp, xyl, and bph operons . Although the product of ORF12 was found to exhibit HOMODA and 2-hydroxy-6-oxohepta-2,4-dienoic acid (HOHDA) hydrolase activities, it did not exhibit 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase activity . The deduced amino acid sequence of ORF11 showed 40.4% homology with the sequence of todX in Pseudomonas putida F1 (Y . Wang, M . Ralings, D . T . Gibson, D . Labbe, H . Bergeron, R . Brousseau, and P . C . K . Lau, Mol . Gen . Genet . 246:570-579, 1995) . The nucleotide sequence of ORF13 and its flanking region showed strong homology (91.0%) with IS52 from Pseudomonas savastanoi (Y . Yamada, P.-D . Lee, and T . Kosuge, Proc . Natl . Acad . Sci . USA 83:8263-8267, 1982) . By characterization of cumH and cumD, the entire cum gene cluster from the cumene-degrader P . fluorescens IP01 (cumA1A2A3A4BCEGFHD) has been identified.

Mikrobiologiia, 1996 Nov-Dec, 65(6), 813 - 7
{Production of indole-3-acetic acid by rhizosphere bacteria of the genus Pseudomonas during the growth process}; Oliunina LN et al.; The production of indole-3-acetic acid (IAA) by batch cultures of the rhizosphere bacteria Pseudomonas fluorescens 20 and Pseudomonas putida 23, known to stimulate plant growth, was studied by immunoenzymatic analysis and the Gordon-Weber method . The accumulation of IAA exhibited two maxima, in the 9th and 40th hours of cultivation . In both pseudomonad cultures, the first maximum occurred in the exponential phase, and the second maximum corresponded to the beginning of the stationary growth phase . Both maxima of IAA accumulation coincided with decreases in the specific growth rates of the cultures . The stationary-phase culture of P . putida 23, as compared to that of P . fluorescens 20, was characterized by greater IAA content, which was retained for a long time.

Steroids, 1996 Nov, 61(11), 627 - 33
Sterol peroxidation by Pseudomonas fluorescens cholesterol oxidase; Teng JI et al.; Cholesterol is oxidized by commercially available Pseudomonas fluorescens cholesterol oxidase to 6 beta-hydroperoxycholest-4-en-3-one as the initial product, with none of the expected produce, cholest-4-en-3-one, formed . The transformation indicates that P . fluorescens cholesterol oxidase also acts as a flavoprotein dioxygenase.

Biochem J, 1996 Oct 15, 319 ( Pt 2), 515 - 20
Evidence that linker sequences and cellulose-binding domains enhance the activity of hemicellulases against complex substrates; Black GW et al.; Xylanase A (XYLA) and arabinofuranosidase C (XYLC) from Pseudomonas fluorescens subsp . cellulosa are modular enzymes consisting of discrete cellulose-binding domains (CBDs) and catalytic domains joined by serine-rich linker sequences . To evaluate the role of the CBDs and interdomain regions, the capacity of full-length and truncated derivatives of the two enzymes, lacking either the linker sequences or CBDs, to hydrolyse a range of substrates, and bind to cellulose, was determined . Removal of the CBDs did not affect either the activity of XYLA or XYLC against soluble arabinoxylan . Similarly, deletion of the linker sequences did not alter the affinity of the enzymes for cellulose or their activity against soluble substrates, even when bound to cellulose via the CBDs . Truncated derivatives of XYLA lacking either the linker sequences or the CBD were less active against xylan contained in cellulose-hemicellulose complexes, compared with the full-length xylanase . Similarly, removal of the CBD from XYLC diminished the activity of the enzyme (XYLC''') against plant-cell-wall material containing highly substituted arabinoxylan . The role of CBDs and linker sequences in the catalytic activity of hemicellulases against the plant cell wall is discussed.

FEMS Microbiol Lett, 1996 Oct 15, 144(1), 61 - 6
Regulation of the iron uptake genes in Pseudomonas fluorescens M114 by pseudobactin M114: the pbrA sigma factor gene does not mediate the siderophore regulatory response; Callanan M et al.; The iron-regulated PbrA sigma factor dictates the production of the siderophore, pseudobactin M114, and its cognate outer membrane receptor, PbuA, in Pseudomonas fluorescens M114 . However, the siderophore molecule also has a role in regulating the expression of the siderophore biosynthetic and siderophore receptor genes in P . fluorescens M114 . This is based on the fact that beta-galactosidase levels from lacZ fusions of M114 siderophore promoters (biosynthetic and receptor) were reduced in M114 siderophore biosynthetic mutants compared to wild-type M114 . Expression of both promoters was increased by the addition of pseudobactin M114 to the growth medium . This effect was widespread and applicable to all but one of the siderophore negative strains of M114 tested . Furthermore, it was demonstrated that transcription of the pbr A sigma factor gene was not reduced in the siderophore biosynthetic mutants . This excludes the possibility that reduced expression of the siderophore biosynthetic and receptor promoters in the siderophore biosynthetic mutants is mediated at the level of expression of the pbr A gene itself . In addition, it was noted that the siderophore regulated response was applicable to promoters with and without the DNA sequence motif, (G/C)CTAAATCCC, which is required for iron-regulated expression of some pseudomonad promoters.

J Endod, 1996 Oct, 22(10), 535 - 9
Bacterial leakage in endodontics: an improved method for quantification; Michailesco PM et al.; A new method for studying leakage of root fillings using the bacterium Pseudomonas fluorescens ATCC 13525 is described . The presence of the microorganism is detected by fluorimetry and can thus be used to measure the depth of penetration from the root apex toward the crown of the tooth . This system, applied to a number of methods of root canal filling, showed that procedures involving compaction of the gutta-percha gave a more effective seal than the use of a paste sealer with uncondensed gutta-percha . There was no statistically significant difference between the leakage results from the lateral, vertical, and thermomechanical condensed techniques.

FEMS Microbiol Lett, 1996 Oct 1, 143(2-3), 223 - 8
Aluminum detoxication mechanism in Pseudomonas fluorescens is dependent on iron; Appanna VD et al.; The soil microbe Pseudomonas fluorescens has been shown to detoxify aluminum by the elaboration of a soluble metabolite where the trivalent metal is sequestered {Appanna and St . Pierre, FEMS Microbiol . Lett . 24 (1994) 327-332} . The inclusion of 5 mM iron in the growth medium elicited an entirely disparate detoxification strategy . In this instance, the two trivalent metals were immobilized in a gelatinous lipid-rich residue . Dialysis and ultracentrifugation studies indicated that the test metals were being transformed from early stages of growth and were associated with phosphatidylethanolamine . However, at 45 h of cellular multiplication, most of the metals were deposited as an insoluble residue . X-ray fluorescence analyses identified the constituents of this mineral essentially as aluminum, iron and phosphorus . Scanning electron microscopy and energy dispersive X-ray microanalysis of the dialysate, isolated at 35 h of microbial growth, revealed thread-like structures associated with nodule-like bodies that were rich in the two test metals . Transmission electron microscopic studies aided in the visualization of iron and aluminum inclusions within the bacterial cells.

Analyst, 1996 Oct, 121(10), 1451 - 6
Biodegradation studies of selected hydrocarbons from diesel oil; Sepic E et al.; In-vitro biodegradation of aliphatic and aromatic hydrocarbons present in diesel oil by Pseudomonas fluorescens, Texaco was studied in an aqueous medium . Small aliquots of diesel oil and its aromatic fraction were incubated aerobically for periods of up to seven months and analysed by GC-MS . Biotic losses proved to be greater for aliphatic than aromatic compounds . Most biodegradation occurred within the first 20 d of incubation . The most rapid biodegradation, up to 65% in 8 d, was observed for n-alkanes (C14-C18) . The same compounds were also shown to be less affected by abiotic losses . Biodegradation of n-alkanes from diesel oil and diesel oil itself showed first order kinetics for the initial incubation period . Aromatic compounds proved to be resistant to biodegradation and only phenanthrene had been degraded (30%) within 6 months.

J Bacteriol, 1996 Oct, 178(19), 5627 - 35
The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product; MacGregor CH et al.; The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced . Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph) . New crc mutants were constructed by disruption of the wild-type crc gene . The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes . However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains . The crc gene product was overexpressed in both P . aeruginosa and in E . coli, and the Crc protein was purified from both . The purified Crc proteins show neither apurinic/apyrimidinic endonuclease nor exonuclease activity . Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species.

Mol Microbiol, 1996 Sep, 21(6), 1239 - 52
The general L-amino acid permease of Rhizobium leguminosarum is an ABC uptake system that also influences efflux of solutes; Walshaw DL et al.; A general L-amino acid permease (Aap) from the ABC transporter family, encoded by four genes (aapJ, Q, M, P), has been cloned and characterized in Rhizobium leguminosarum . It transports a wide range of L-amino acids but has a preference for those with polar side-chains . A single binding protein of broad specificity (AapJ) is required for transport of all solutes . Unusually for an ABC transporter, Aap has both high affinity for and supports high rates of solute uptake . Genes for putative amino acid permeases with broad specificity for amino acids also exist in Escherichia coli and probably in Pseudomonas fluorescens, although the permease from E . coli does not appear to be expressed . Aap is an active uptake system that also affects the efflux of a broad range of amino acids . Efflux can be measured both as the loss of an intracellular amino acid after the addition of an excess of a homologous or heterologous amino acid, and as excretion of intracellularly synthesized glutamate . Mutation of Aap prevented efflux of intracellular amino acids caused by the addition of an extracellular heterologous amino acid, while overexpression increased the rates of such efflux . Furthermore, excretion of glutamate synthesized inside the cell was reduced by 76% in an aap strain . All four gene products, including the binding protein (AapJ), appear to be needed for efflux . Aap from R . leguminosarum expressed in E . coli also promoted efflux on addition of an extracellular heterologous amino acid . These results indicate either that Aap regulates an efflux channel/transporter or that solute has access to the translocation pathway of Aap from both sides of the membrane.

Biotechnol Prog, 1996 Sep-Oct, 12(5), 718 - 22
Effects of growth rate on the production of Pseudomonas fluorescens lipase during the fed-batch cultivation of Escherichia coli; Kim SS et al.; Recombinant Escherichia coli was grown to produce the thermostable Pseudomonas fluorescens lipase by controlling the specific growth rate (mu) . At low growth rate (mu = 0.07 h-1), a higher cell density (OD600 = 140) was obtained . By calculating the cell yield on nitrogen, the medium was optimized to prevent inhibition of cell growth by nitrogen unbalance . Acetate accumulated in the broth at the same rate as observed in a chemostat under nitrogen-limited fed-batch conditions, but not in a glucose-limited fed-batch culture . Postinduction growth rate affected lipase production more than preinduction growth rate . After induction, more lipase was produced at a low growth rate (mu = 0.1 h-1) than at a high growth rate (mu = 0.4 h-1) . More lipase was produced at 37 than at 20 degrees C at a constant growth rate, indicating that temperature affected other factors in addition to growth rate.

Appl Environ Microbiol, 1996 Sep, 62(9), 3319 - 24
Evidence for two domains of growth temperature for the psychrotrophic bacterium Pseudomonas fluorescens MF0; Guillou C et al.; The variations in the maximal specific growth rate of the psychrotrophic bacterium Pseudomonas fluorescens MF0 with respect to temperature were studied between 0 and 30 degrees C (optimal for growth) . The Arrhenius plot showed a drastic change in slope at the intermediate temperature of 17 degrees C . Over the cold domain from 0 to 17 degrees C, the temperature characteristic was twofold higher than over the suboptimal domain from 17 to 30 degrees C . The macromolecular composition of exponentially growing cells was invariant over the entire range from 0 to 30 degrees C . Variations of temperature and growth rate were independently investigated through chemostat experiments in order to characterize their respective effects on cell macromolecular composition and size . The effect of growth rate in this psychrotrophic strain is identical to that of all other bacteria assayed so far . In contrast, an original biphasic variation of total protein concentration was demonstrated in strain MF0 with respect to temperature, with a maximum at 17 to 20 degrees C . Indeed, increasing the temperature in the chemostat resulted in a biphasic decrease in the net protein production rate: a very slight decrease below 17 degrees C and a much larger decrease from 17 to 28 degrees C . These results could signify an increase in the cellular protein degradation rate with increasing temperature, especially above 17 degrees C.

Appl Environ Microbiol, 1996 Sep, 62(9), 3277 - 83
Inactivation of Aeromonas hydrophila by Fe(II)-related-radical generation in oxidizing groundwaters; Kersters I et al.; The survival of Aeromonas hydrophila AWWX1 in filter-sterilized phreatic groundwaters was studied by using viable counts . Aeromonas counts rapidly decreased 2 to 3 log units in oxidizing raw groundwaters from Snellegem and Beernem, Belgium (Snellegem-raw and Beernem-raw, respectively), containing high concentrations of Fe2+ (460 to 1,070 microM) . The rapid decline in viable counts of Aeromonas cells in the oxidizing raw groundwater of Snellegem was prevented by the addition of an Fe2+ chelator (2,2'-dipyridyl) or compounds (i.e., ascorbic acid and catalase) that act on toxic oxygen species . The results suggest that free radicals, generated spontaneously in oxidizing Fe2+-containing groundwaters, caused the inactivation of A . hydrophila AWWX1 . Evidence that free radicals are generated under the given conditions was provided by the observation that propylphosphonic acid, a compound which is very susceptible to radicals, was degraded upon addition to these waters . A . hydrophila PWBS, Pseudomonas fluorescens P17, Spirillum strain NOX, and heterotrophs showed decreases in culturability in filter-sterilized Snellegem-raw water similar to that shown by A . hydrophila AWWX1 . These findings indicate that free radicals generated in Fe2+-containing groundwaters upon aeration are capable of inactivating various bacterial species.

Biodegradation, 1996 Aug, 7(4), 353 - 66
Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens; Foght JM et al.; Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources . The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites . A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P . fluorescens LP6a . This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns . In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene . Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes . Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis . Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.

Mol Microbiol, 1996 Aug, 21(4), 777 - 85
A cytochrome c biogenesis gene involved in pyoverdine production in Pseudomonas fluorescens ATCC 17400; Gaballa A et al.; Pseudomonas fluorescens ATCC 17400 produces pyoverdine under iron-limiting conditions . A Tn5 mutant, 2G11, produced lower amounts of different pyoverdine forms and was unable to grow under iron limitation caused by ethylenediamine-di(o-hydroxy-phenylacetic acid) (EDDHA) or zinc . This mutant was complemented by a 9.6 kb HindIII-BamHI DNA fragment that contained eight contiguous open reading frames (ORFs cytA to cytH) . The proteins possibly encoded by this polycistronic gene cluster were all similar to the products of cytochrome c biogenesis genes from, amongst others, Rhodobacter capsulatus and Bradyrhizobium japonicum, not only in terms of amino acid sequence, but also in the overall hydropathy index of these proteins . By TnphoA mutagenesis and site-specific gene replacement it was found that the first three ORFs (cytA to cytC) were essential for cytochrome c production while only the product of cytA was needed for normal pyoverdine production . The presence of a putative haem-binding site in the CytA protein (WGSWWVWD) was confirmed . From analysis of a constructed phoA fusion, a periplasmic location was found for this motif . The ability of the cytA gene to restore both cytochrome c and pyoverdine production suggests the involvement of this particular gene both in haem and in pyoverdine transport in P . fluorescens.

Optom Vis Sci, 1996 Aug, 73(8), 529 - 32
Bacterial contamination of hydrophilic contact lens solutions marketed in spain; Durban JJ et al.; Bacterial contamination of hydrophilic contact lens solutions may play an important role in contact lens-associated ocular infections . This study investigated bacterial contamination in 52 different hydrophilic contact lens solutions marketed in Spain by 12 different companies . We filtered the entire contents of 5 new, factory-sealed bottles from each of the 52 brands and cultured the fitter on a neutralizing broth plate . Bacteria were cultured, isolated, and identified from 29 of the 260 bottles tested (11.15%) . Eight of the 52 brands had at least 1 of the 5 bottles contaminated (15.38%) . Contaminated solutions originated from four different companies . One manufacturer contributed most of the positive cases due, presumably, to an industrial contamination by Pseudomonas fluorescens . The rest of the culture-positive bottles were contaminated by Bacillus spp . and Oerskovia spp.

J Antibiot (Tokyo), 1996 Aug, 49(8), 758 - 64
Kanchanamycins, new polyol macrolide antibiotics produced by Streptomyces olivaceus Tü 4018 . I . Taxonomy, fermentation, isolation and biological activities; Fiedler HP et al.; The kanchanamycins, a group of novel 36-membered polyol macrolide antibiotics were detected in the culture filtrate and mycelium of Streptomyces olivaceus Tu 4018 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening . The compounds show antibacterial and antifungal activities, and are especially effective against Pseudomonas fluorescens . Besides the kanchanamycin complex, strain Tu 4018 produces the 42-membered macrolactones, oasomycin A and desertomycin A, as well as tryptophan-dehydrobutyrine diketopiperazine and daidzein.

Microbiology, 1996 Aug, 142 ( Pt 8), 2129 - 35
The non-haem chloroperoxidase from Pseudomonas fluorescens and its relationship to pyrrolnitrin biosynthesis; Kirner S et al.; The non-haem chloroperoxidase gene (cpoF) from the pyrrolnitrin producer Pseudomonas fluorescens BL914 was cloned using an oligonucleotide derived from part of the N-terminal amino acid sequence of chloroperoxidase (CPO-P) from Pseudomonas pyrrocina as a probe . Based on the overexpression of cpoF in Escherichia coli and the stability of CPO-F against higher temperatures and proteases, the enzyme was purified to homogeneity . Partial characterization of the enzyme showed that it belongs to the class of bacterial non-haem CPOs . To investigate the role of CPO-F in pyrrolnitrin biosynthesis, the cpoF gene was inactivated by insertion of a kanamycin cassette . Exchange of the chromosomal cpoF gene against the disrupted copy had no influence on pyrrolnitrin production demonstrating that CPO-F was not involved in pyrrolnitrin biosynthesis.

J Biotechnol, 1996 Jul 18, 48(1-2), 129 - 36
Cellular response to a multiple-metal stress in Pseudomonas fluorescens; Appanna VD et al.; Pseudomonas fluorescens multiplied in a minimal mineral medium supplemented with millimolar amounts of aluminum (5 mM), iron (5 mM), zinc (3 mM), calcium (2 mM) and gallium (1 mM) . A slight decrease in growth rate and a 22% diminution in cellular yield were observed as compared to the control medium . Citrate, the sole source of carbon to which the test metals were complexed, was completely utilized . Although at stationary phase of growth most of the metals were immobilized in an exocellular lipid-rich residue, ultracentrifugation and dialysis studies revealed that metals were associated with phosphatidylethanolamine (PE) from early stages of growth . As growth progressed the metal content of the soluble cellular extract increased reaching an optimum at 35 h of incubation . However, no detectable amounts of metals in this cellular component were discerned at stationary phase of growth . There appeared to be no marked variation in exocellular protein and carbohydrate production in control and metal-stressed cultures . Transmission electron microscopic studies revealed metal rich bodies associated with the cytoplasm . Scanning electron microscopic analyses of the dialyzate aided in the identification of the metal-rich bodies associated with elongated structures comprised of carbon, oxygen and phosphorus . PE appeared to be an important organic constituent of the gelatinous residue.

Eur J Biochem, 1996 Jul 15, 239(2), 469 - 78
4-Hydroxybenzoate hydroxylase from Pseudomonas sp . CBS3 . Purification, characterization, gene cloning, sequence analysis and assignment of structural features determining the coenzyme specificity; Seibold B et al.; 4-Hydroxybenzoate hydroxylase from Pseudomonas sp . CBS3 was purified by five consecutive steps to apparent homogeneity . The enrichment was 50-fold with a yield of about 20% . The enzyme is a homodimeric flavoprotein monooxygenase with each 44-kDa polypeptide chain containing one FAD molecule as a rather weakly bound prosthetic group . In contrast to other 4-hydroxybenzoate hydroxylases of known primary structure, the enzyme preferred NADH over NADPH as electron donor . The pH optimum for catalysis was pH 8.0 with a maximum turnover rate around 45 degrees C . Chloride ions were inhibitory, and competitive with respect to NADH . 4-Hydroxybenzoate hydroxylase from Pseudomonas sp . CBS3 has a narrow substrate specificity . In addition to the transformation of 4-hydroxybenzoate to 3,4-dihydroxybenzoate, the enzyme converted 2-fluoro-4-hydroxybenzoate, 2-chloro-4-hydroxybenzoate, and 2,4-dihydroxybenzoate . With all aromatic substrates, no uncoupling of hydroxylation was observed . The gene encoding 4-hydroxybenzoate hydroxylase from Pseudomonas sp . CBS3 was cloned in Escherichia coli . Nucleotide sequence analysis revealed an open reading frame of 1182 bp that corresponded to a protein of 394 amino acid residues . Upstream of the pobA gene, a sequence resembling an E . coli promoter was identified, which led to constitutive expression of the cloned gene in E . coli TG1 . The deduced amino acid sequence of Pseudomonas sp . CBS3 4-hydroxybenzoate hydroxylase revealed 53% identity with that of the pobA enzyme from Pseudomonas fluorescens for which a three-dimensional structure is known . The active-site residues and the fingerprint sequences associated with FAD binding are strictly conserved . This and the conservation of secondary structures implies that the enzymes share a similar three-dimensional fold . Based on an isolated region of sequence divergence and site-directed mutagenesis data of 4-hydroxybenzoate hydroxylase from P . fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7315 - 20
A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens; Yang CH et al.; A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil . Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome . The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria . Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme . Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c . However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper.

Gig Sanit, 1996 Jul-Aug, (4), 8 - 10
{Hygienic evaluation of combined biotechnology for treating sewage from artificial accumulations}; Rozvaga RI et al.; Combined biotechnology for treating sewage and industrial wastes with Pseudomonas fluorescens bacteria and Scirpus lacustris L . reed mass was developed . The treated waters can be discharged into the surface reservoirs.

Appl Environ Microbiol, 1996 Jul, 62(7), 2264 - 72
Comparison of methods for detection and enumeration of airborne microorganisms collected by liquid impingement; Terzieva S et al.; Bacterial agents and cell components can be spread as bioaerosols, producing infections and asthmatic problems . This study compares four methods for the detection and enumeration of aerosolized bacteria collected in an AGI-30 impinger . Changes in the total and viable concentrations of Pseudomonas fluorescens in the collection fluid with respect to time of impingement were determined . Two direct microscopic methods (acridine orange and BacLight) and aerodynamic aerosol-size spectrometry (Aerosizer) were employed to measure the total bacterial cell concentrations in the impinger collection fluid and the air, respectively . These data were compared with plate counts on selective (MacConkey agar) and nonselective (Trypticase soy agar) media, and the percentages of culturable cells in the collection fluid and the bacterial injury response to the impingement process were determined' . The bacterial collection rate was found to be relatively unchanged during 60 min of impingement . The aerosol measurements indicated an increased amount of cell fragments upstream of the impinger due to continuous bacterial nebulization . Some of the bacterial clusters, present in the air upstream of the impinger, deagglomerated during impingement, thus increasing the total bacterial count by both direct microscopic methods . The BacLight staining technique was also used to determine the changes in viable bacterial concentration during the impingement process . The percentage of viable bacteria, determined as a ratio of BacLight live to total counts was only 20% after 60 min of sampling . High counts on Trypticase soy agar indicated that most of the injured cells could recover . On the other hand, the counts from the MacConkey agar were very low, indicating that most of the cells were structurally damaged in the impinger . The comparison of data on the percentage of injured bacteria obtained by the traditional plate count with the data on percentage of nonviable bacteria obtained by the BacLight method showed good agreement.

Plant Cell, 1996 Jul, 8(7), 1095 - 105
Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific hypersensitive cell death; Gopalan S et al.; The nonpathogenic bacteria Pseudomonas fluorescens and Escherichia coli can elicit a genotype-specific hypersensitive response (HR) in plants if they express both the HR and pathogenesis (Hrp) protein secretion system and the HrpZ harpin from P . syringae pv syringae 61 and a P . syringae avirulence (avr) gene whose presence is recognized by a corresponding disease resistance gene in the plant . We have found that the recognition event appears to require transfer of the Avr protein into the plant cell . Elicitation of a genotype-specific HR was observed with avrB+ P . fluorescens in soybean and Arabidopsis plants carrying resistance genes RPG1 and RPM1, respectively, and with avrPto+ E . coll in tomato plants carrying resistance gene PTO, but only if the Hrp secretion system, HrpZ, and the appropriate Avr proteins were produced in the same bacterial cell . The failure of avrB hyperexpression and exogenous AvrB or HrpZ to alleviate these requirements in soybean and Arabidopsis suggests that the site of AvrB action is not in the bacterial cell or plant apoplast . An Arabidopsis rps3 (rpm1) glabrous1 mutant was transformed with constructs expressing avrB and was crossed with an Arabidopsis ecotype Columbia (RPM1 GLABROUS1) plant . F1 seedlings (identified by their kanamycin-resistant, pubescent phenotype) exhibited extensive necrosis on cotyledon leaves 10 days postgermination . Ecotype Columbia and rps3-1 leaves biolistically cobombarded with plasmids expressing the beta-glucuronidase (GUS) gene and avrB failed to produce GUS activity (indicative of cell death) only when RPM1 and avrB were present in the leaf . Thus, both stable and transient expression of avrB in Arabidopsis resulted in RPM1-dependent necrosis, and the only demonstrable site of action for AvrB was inside plant cells.

J Appl Bacteriol, 1996 Jul, 81(1), 1 - 6
Cross-reactivity of antibodies raised to Pseudomonas fluorescens protease with extracellular proteins produced by meat-spoiling pseudomonads; Lundy FT et al.; The cross-reactivity patterns of antibodies to Pseudomonas fluorescens protease with the extracellular proteins produced by a number of meat-spoiling pseudomonads were studied . Immunoblotting studies showed that purified IgG to Ps . fluorescens protease cross-reacted with extracellular proteins in the cell culture supernatant fluids of Pseudomonas spp., including Ps . fragi and Ps . lundensis . In the case of Ps . lundensis and Pseudomonas spp . 11390, the cross-reactive moieties were of similar molecular weight to the Ps . fluorescens protease (46 kDa) . However, in Ps . fragi the cross-reactive moiety was a lower molecular weight protein (8 kDa) . This may represent a fragment of the active enzyme . These results indicate the presence of common antigenic determinants among the proteases of meat spoiling pseudomonads.

Biol Pharm Bull, 1996 Jun, 19(6), 873 - 5
Biochemical characterization of a Pseudomonas fluorescens strain isolated from a benzalkonium chloride solution; Nagai K et al.; A bacterium isolated as the contaminant of a batch of commercial benzalkonium chloride (BAC) solution (10% (w/v)) stored in a loosely capped bottle in the Department of Pharmacy Shinshu University Hospital was identified as Pseudomonas fluorescens belonging to biotype G of Stanier, et al . The strain was highly resistant to BAC, and the lowest concentration of BAC that inhibited visible growth of the strain as measured on nutrient agar plates was > or = 5000 micrograms/ml . BAC is a typical quaternary ammonium detergent . Thus we examined the tolerable growth concentration of various strains on surfactants . We were able to confirm growth of P . fluorescens of BAC resistance strain (PFRB) in 5% concentration, but the other strains were not able to grow in 0.1% concentration . We investigated the relationship between biotype and resistance to BAC . PFRB and three clinical isolated strains were found to be the same biotype G . However, no apparent correlation was found between the same biotypes and minimum inhibitory concentration (MIC) of disinfectant or growth permissible concentration on surfactants . The strain was unable to decompose BAC, as no growth occurred in the minimum medium containing BAC as the sole source of carbon, nitrogen or both . Our finding caused us to realize that P . fluorescens might also be a contaminant of disinfectants, as we have seen in Pseudomonas cepacia.

J Bacteriol, 1996 Jun, 178(11), 3308 - 13
Mutational analysis of the active site of Pseudomonas fluorescens pyrrolidone carboxyl peptidase; Le Saux O et al.; On the basis of chemical inhibition studies and a multiple alignment of four pyrrolidone carboxyl peptidase (Pcp) amino acid sequences, seven conserved residues of the Pseudomonas fluorescens Pcp, which might be important for enzyme activity, have been modified by site-directed mutagenesis experiments . Wild-type and mutant Pcps were expressed in Escherichia coli, purified, and characterized by the ability to cleave the synthetic chromogenic substrate pyroglutamyl-beta-naphthylamide and the dipeptide pyroglutamyl-alanine . Substitution of Glu-10 and Glu-22 by Gln led to enzymes which displayed catalytic properties and sensitivities to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide similar to those of the wild-type Pcp . These residues are not essential for the catalytic activity . Replacement of Asp-89 by Asn and Ala resulted in enzymes which retained nearly 25% of activity and which had no activity, respectively . Substitution of the Cys-144 and His-166 residues by Ala and Ser, respectively, resulted in inactive enzymes . Proteins with changes of Glu-81 to Gln and Asp-94 to Asn were not detectable in crude extract and were probably unstable in bacteria . Our results are consistent with the proposal that Cys-144 and His-166 constitute the nucleophilic and imidazole residues of the Pcp active site, while residue Glu-81, Asp-89, or Asp-94 might constitute the third part of the active site . These results lead us to propose Pcps as a new class of thiol aminopeptidases.

Mikrobiologiia, 1996 May-Jun, 65(3), 318 - 25
{Characterization of the lipopolysaccharide from the Pseudomonas fluorescens strain IMV 472 (biovar I)}; Veremeichenko SN et al.; Lipopolysaccharide was isolated from dry biomass of the Pseudomonas fluorescens strain IMV 472 (biovar I) by the Westphal procedure and purified by ultracentrifugation . Fractions of the structural components of the lipopolysaccharide macromolecule-lipid A, the core oligosaccharide, and the O-chain-were isolated and characterized . 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, hexadecenoic, octadecenoic, and hexadecanoic acids were identified as the main fatty acids of lipid A . The hydrophilic moiety of lipid A was found to include glucosamine, phosphoethanolamine, and phosphate . Glucose, galactose, rhamnose, glucosamine, galactosamine, alanine, phosphoethanolamine, and 2-keto-3-deoxyoctulosonic were identified in the core region of lipid A . The O-specific polysaccharide chain consisted of a repeating tetrasaccharide fragment that included two L-rhamnose residues, a D-fucose residue, and an N-acetyl-D-glucosamine residue as a side substituent . Double immunodiffusion in agar and the lipopolysaccharide precipitation reaction revealed no serological interrelation between strain IMV 472 and P . fluorescens strains studied previously.

Plant Mol Biol, 1996 May, 31(2), 255 - 65
Characterization of hsr201 and hsr515, two tobacco genes preferentially expressed during the hypersensitive reaction provoked by phytopathogenic bacteria; Czernic P et al.; During an incompatible interaction between tobacco and the bacterial phytopathogen Pseudomonas solanacearum, 2 classes of genes, the so-called hsr (hypersensitivity-related) genes, activated preferentially during the hypersensitive reaction, and the str (sensitivity-related) genes, expressed strongly during compatible and incompatible interactions, have been identified . In this report, two hsr cDNA clones, hsr515 and hsr201, as well as their expression patterns are presented . Hsr515 was found to encode a P450 monooxygenase and is most similar to the ripening-related avocado gene CYP71A1 (40.6% amino acid identity) . Hsr201 presents 58.6% amino acid identity with pTom36, a tomato gene expressed during fruit maturation . The putative functions of the hsr gene products appear to be quite diverse and their characteristics of activation were found to be very conserved: accumulation of the corresponding mRNAs primarily in leaf areas in contact with the avirulent P . solanacearum strain or with a Pseudomonas fluorescens strain containing the hrpZ gene encoding a necrotizing polypeptide, harpin and absence of expression during normal plant development . Our results also suggest that, in a tobacco line expressing NahG, a lower level of salicylic acid, a compound associated with systemic acquired resistance, and also possibly involved in the development of necrotic lesions characteristic of the HR, does not affect the hsr gene expression.

Can J Microbiol, 1996 May, 42(5), 446 - 52
Use of continuous culture to screen for lipase-producing microorganisms and interesterification of butter fat by lipase isolates; Pabai F et al.; The continuous cultivation technique was used to investigate the screening for lipase-producing microorganisms from four commercial starters suitable for the degradation of domestic wastes . Using this technique, three strains of lipase-producing bacteria were isolated and identified: Pantoea agglomerans (BB96CC1, BB168CC2) and Pseudomonas fluorescens (BW96CC1) . In addition, butter fat induced more lipase production when present in the growth medium . Interesterification of butter fat triacylglycerols by enzymatic extracts of the isolated strains of microorganisms resulted in an appreciable interesterification yield, implying that hydrolysis was suppressed and interesterification of butter fat triacylglycerols was maximized in a microemulsion free-cosurfactant system.

Am Ind Hyg Assoc J, 1996 Apr, 57(4), 348 - 55
Penetration of airborne microorganisms through a surgical mask and a dust/mist respirator; Willeke K et al.; This study investigated bacterial penetration of different bacterial shapes, aerodynamic sizes, and flow rates through a surgical mask and a dust/mist respirator . The bacterial penetrations were compared with those of spherical corn oil particles of the same aerodynamic diameter tested under the same conditions . The tests were performed at different levels of aerosol penetration . Bacteria, ranging from spherical to rod-shaped with a high aspect (length to width) ratio, were selected as test agents . Among these, Pseudomonas fluorescens physically simulates Mycobacterium tuberculosis by shape and size . The concentrations of bacteria upstream and downstream of the test devices were measured with an aerodynamic size spectrometer . This instrument was found to measure the total viable and nonviable bacterial concentration effectively and dynamically over the entire bacterial size range down to 0.5 microns in aerodynamic size . The results indicate that the spherical corn oil particles and the spherical Streptococcus salivarius bacteria have the same penetration in the size range from 0.9 to 1.7 microns . It has been found that rod-shaped bacteria penetrate less . The penetration difference between the spherical and rod-shaped bacteria depends on the aspect ratio of the bacteria . For an aspect ratio of 4, the penetration of rod-shaped bacteria is about half that of spherical ones . Thus, it is projected that a respirator with 90% efficiency against spherical microorganisms or test particles (10% penetration) will be 95% efficient against rod-shaped microorganisms of the same aerodynamic equivalent diameter with an aspect ratio of 3 to 4, such as Mycobacterium tuberculosis (5% penetration).

Avian Dis, 1996 Apr-Jun, 40(2), 473 - 6
Necrotizing hepatitis in pet birds associated with Pseudomonas fluorescens; Jackson MK et al.; Six pet birds, from a flock of 100 birds of various species, died within a 2-day period . Drinking water had recently been changed from potable water to irrigation water . Three birds submitted for necropsy had hepatic necrosis with numerous gram-negative rodshaped bacteria present in necrotic areas and Kuppfer cells . Pseudomonas fluorescens was isolated in pure culture from the livers of all three birds and from other organs . This is the first report of naturally occurring disease in which P . fluorescens was the sole etiologic agent identified.

New Microbiol, 1996 Apr, 19(2), 113 - 21
Characterisation of a phospholipase C produced by Pseudomonas fluorescens; Ivanov A et al.; Phospholipase C (phosphatidylcholine phosphohydrolase, EC 3.1.4.3) and lipase (EC 3.1.1.3) activities were detected in the supernatant fluid of Pseudomonas fluorescens strain D cultures . A combination of ultrafiltration and successive chromatography through columns of Sephadex G-75 and DEAE-cellulose was used to purify the phospholipase C over 700-fold from the culture medium, with 28.5% yield . The purified enzyme appeared as a single band after polyacrylamide gel electrophoresis . The apparent molecular mass of the phospholipase C was 36,000 daltons when estimated by gel permeation chromatography . The purified enzyme hydrolysed phosphatidylcholine more efficiently than phosphatidylethanolamine . The synthetic substrate p-nitrophenylphosphorylcholine, phosphatidylinositol or sphingomyelin were not hydrolysed . Hydrolysis of phosphatidylcholine was inhibited by EDTA (1mM) and stimulated by Zn2+, Mg2+ ions and detergents . These properties of the enzyme indicate that it is distinct from the previously reported Ps . fluorescens phospholipase C.

Carbohydr Res, 1996 Mar 22, 283, 129 - 39
Structure of a decasaccharide isolated by mild acid degradation and dephosphorylation of the lipopolysaccharide of Pseudomonas fluorescens strain ATCC 49271; Knirel YA et al.; Mild acid degradation of the Pseudomonas fluorescens strain ATCC 49271 lipopolysaccharide resulted in a core oligosaccharide containing D-glucose, 2-acetamido-2-deoxy-D- glucose, 2-(L- alanylamino)-2-deoxy-D-galactose, 2-acetamido-2,6-dideoxy-D-glucose (QuiNAc), 2-acetamido- 2,6-dideoxy-L-galactose (FucNAc), L-glycero-D-manno-heptose (Hep), 3-deoxy-D- manno-octulosonic acid (Kdo, present in multiple forms), and 5-acetamidino-7-acetamido-3,5,7,9- tetradeoxy- L-glycero-D-galacto-nonulosonic acid (a di-N-acyl derivative of legionaminic acid, Non) as well as O-acetyl, O-carbamoyl, and phosphate groups, including triphosphate groups . The dephosphorylated (HF) decasaccharide and products of its partial and full O-deacylation were studied by methylation analysis, GLC-MS, and 1H NMR spectroscopy, including 1D NOE and 2D shift-correlated spectroscopy (COSY) . The core oligosaccharide of P . fluorescens strain ATCC 49271 was found to be a decasaccharide (with partially degraded Kdo region) and one O-antigen repeating unit (di-N-acyllegionaminic acid, Non) attached . The following structure of the dephosphorylated core oligosaccharide was established: {sequence: see text}

Mikrobiol Z, 1996 Mar-Apr, 58(2), 70 - 5
{The determination of phosphoethanolamine and other amino components in the lipopolysaccharides of gram-negative bacteria by ion-exchange chromatography}; Veremeichenko SN; A method for analysis of phosphoethanolamine in hydrolysates of the fractions structural parts of lipopolysaccharide macromolecule on the column with cation-exchange resin Ostion LG ANB (CSFR) in the system of step gradient of sodium-citrate buffers is suggested, optimal conditions for acidic hydrolysis of polysaccharide-standard were chosen to determine phosphoethanolamine and other amino compounds contained in it . The conditions for chromatography analysis of phosphoethanolamine together with aminosugars and amino acids have been developed . Using the suggested procedure the presence of phosphoethanolamine was found in preparations of lipid A hydrolysates and core oligosaccharide isolated from some lipopolysaccharide strains of Pseudomonas fluorescens.

J Appl Bacteriol, 1996 Mar, 80(3), 266 - 76
Survival potential of Aeromonas hydrophila in freshwaters and nutrient-poor waters in comparison with other bacteria; Kersters I et al.; The survival of a genetically-marked Aeromonas hydrophila strain was studied in water microcosms using viable counts . Aeromonas hydrophila AWWX1 was shown to survive without decline in viable counts for at least 10 d in three of four filtered-autoclaved freshwaters (surface water and groundwater) and in all examined filtered-autoclaved nutrient-poor waters (bottled spring water, Milli-Q and tap water) . However, in the unfiltered waters, a rapid decrease in viable counts of Aer . hydrophila AWWX1 was observed after 1-5 d . The survival of Aer . hydrophila AWWX1 in nutrient-poor waters was compared with that of Pseudomonas fluorescens P17 and Spirillum strain NOX . Survival characteristics were organism- and water-dependent . In the filtered-autoclaved waters, viable counts of Spirillum strain NOX were ca 1 log-unit higher than for Aer . hydrophila AWWX1 and Ps . fluorescens P17 . The tested strains Aer . hydrophila AWWX1 and Ps . fluorescens P17 survived 3 to 20, respectively 2 to 4 times better in the filtered-autoclaved waters compared to the unfiltered waters . Apparently, any inherent capability of these micro-organisms to adapt to low-nutrient environments was undone by the presence of the autochthonous microbiota . The present findings that Aer . hydrophila survives very poorly in several drinking waters is of utmost importance towards public health and arises questions about the mechanisms involved.

Lett Appl Microbiol, 1996 Mar, 22(3), 214 - 8
Antimicrobial activity of Pseudomonas spp . against food poisoning bacteria and moulds; Laine MH et al.; The present study demonstrates the siderophore production of two strains of Pseudomonas fluorescens and two of Ps . chlororaphis . The antimicrobial activities of these strains were studied against Salmonella typhimurium, Staphylococcus aureus, Fusarium culmorum, F . oxysporum and Aspergillus niger . Despite equal siderophore activities with various Pseudomonas spp . as measured by the chrome azurol S assay, the study shows how siderophore activity does not correlate with the antibacterial activity against food pathogens or with the antimould activity against pathogenic moulds . Furthermore, the results illustrate how siderophores are able to act both as growth inhibitors and stimulators.

Mol Plant Microbe Interact, 1996 Mar, 9(2), 83 - 90
Characterization of a genomic locus required for synthesis of the antibiotic 2,4-diacetylphloroglucinol by the biological control agent Pseudomonas fluorescens Q2-87; Bangera MG et al.; The antibiotic 2,4-diacetylphloroglucinol (Phl) is an important factor in the biological control by fluorescent Pseudomonas spp . of many soilborne diseases including take-all disease of wheat . A 6.5-kb genomic DNA fragment from Pseudomonas fluorescens Q2-87 conferred production of Phl and of a red pigment distinct from Phl, but which typically is present when Phl is produced, upon all of 13 Phl-nonproducing recipient Pseudomonas strains into which it was introduced . Larger fragments that included flanking DNA sequences did not transfer this capability, suggesting that they contain negative regulatory element(s) . Analysis of the 6.5-kb fragment by Tn3HoHo1 mutagenesis further localized the sequences required for Phl production to a segment of approximately 5 kb and revealed the presence of at least two divergently oriented transcriptional units . Insertions within the smaller unit or within about 3 kb of the 5' end of the larger unit caused loss of production of both Phl and the red pigment . Other insertions within the distal 1.5 kb of the larger transcriptional unit abolished production of only the red pigment . Pleiotropic changes in secondary metabolism or colony morphology were not observed in Pseudomonas strains containing the 6.5-kb fragment, although some Phl-producing derivatives grew more slowly and gave rise to smaller colonies than did the wild-type parental strains . The size of the genomic region involved in Phl production, and the consistency and specificity with which these sequences transferred Phl biosynthetic capability, support the conclusion that the 6.5-kb fragment contains the Phl biosynthetic locus.

J Dairy Sci, 1996 Mar, 79(3), 357 - 65
Purification and characterization of a dipeptidase from Pseudomonas fluorescens ATCC 948; Gobbetti M et al.; A dipeptidase from a cell extract of Pseudomonas fluorescens ATCC 948 was purified to homogeneity by ion-exchange chromatography, hydroxyapatite chromatography, gel filtration, and FPLC Phenyl Superose chromatography . The enzyme was located in the cytoplasmic fraction . The dipeptidase appeared to be a monomer with a molecular mass of about 46 kDa, and activity was optimal at pH 8.0 and 50 degrees C . Although activity decreased markedly at temperatures > 50 degrees C, about 50% of maximal activity was retained at 10 degrees C . The activity was inhibited by EDTA, but could be reactivated with Co2+ and partially reactivated with Ca2+ . Reducing agents, such as dithiothreitol and 8-hydroxyquinoline, also strongly inhibited the enzyme . The dipeptidase hydrolyzed only dipeptides and showed a particular aptitude for substrates containing a hydrophobic AA at the N-terminus . Dipeptides containing a proline residue were not cleaved . Kinetic studies indicated that the dipeptidase hydrolyzed Leu-Leu with a Michaelis-Menten constant of 0.43 mM and a turnover number of 1812/s.

J Antibiot (Tokyo), 1996 Mar, 49(3), 263 - 6
Karalicin, a new biologically active compound from Pseudomonas fluorescens/putida . II . Biological properties; Lampis G et al.; The biological activities of karalicin, a new product from the Pseudomonas fluorescens/putida strain SS-3 (CCM 4430) are described . It shows a weak, but specific and irreversible, antiviral activity on Herpes simplex viruses . It also presents some inhibitory activity on different species of yeasts.

J Antibiot (Tokyo), 1996 Mar, 49(3), 260 - 2
Karalicin, a new biologically active compound from Pseudomonas fluorescens/putida . I . Production, isolation, physico-chemical properties and structure elucidation; Lampis G et al.; An original compound, named karalicin, was isolated from a fermentation broth of the Pseudomonas fluorescens/putida strain SS-3 (CCM 4430) . Production, physico-chemical properties and structure elucidation are described.

Appl Microbiol Biotechnol, 1996 Feb, 44(6), 740 - 5
Improved delivery of biocontrol Pseudomonas and their antifungal metabolites using alginate polymers; Russo A et al.; Alginate polymer was evaluated as a carrier for seed inoculation with a genetically modified strain Pseudomonas fluorescens F113LacZY, which protects sugar-beet against Pythium-mediated damping-off . F113LacZY survived in alginate beads at 5 log10 CFU/bead or higher counts for 8 weeks of storage, regardless of the conditions of incubation . In plant inoculation experiments, colonisation of the growing area of the root by F113LacZY, derived from alginate beads placed in the soil next to the seed or from an alginate coating around the seeds, was improved compared with application of just free cells of the strain . F113LacZY trapped in alginate beads was an effective producer of antifungal phloroglucinols as indicated by direct HPLC quantification of phloroglucinols and in vitro inhibition of both the indicator bacterium Bacillus subtilis A1 and the pathogenic fungus Pythium ultimum . Alginate polymer represents a promising carrier for the delivery of biocontrol inoculants for root colonisation and production of antifungal metabolites.

Mol Microbiol, 1996 Feb, 19(3), 521 - 33
Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome; Rainey PB et al.; Pseudomonas fluorescens is a saprophytic bacterium commonly isolated from soil, water, and the surfaces and tissues of plants and animals . The species has important applications in biotechnology because it can enhance plant growth and protect crops against disease . A complete physical map of the 6.63 Mbp P . fluorescens SBW25 chromosome was constructed using data obtained from combinations of one- and two-dimensional electrophoresis of completely or partially digested chromosomal DNA with end labelling . In total, 139 restriction sites (15 PacI, 53 SpeI, 71 XbaI) were placed on the physical map and complete maps of the circular chromosome were obtained for both PacI and SpeI; only XbaI fragments linking SpeI fragments were positioned . The average resolution of restriction sites was 48 kbp . A genetic map was derived from the physical map by southern hybridization and 31 genes were positioned including oriC, rDNA operons (rnnA-E), recA, gacA, and pyvD.

Mol Microbiol, 1996 Feb, 19(4), 715 - 28
Analysis of the role of the Pseudomonas syringae pv . syringae HrpZ harpin in elicitation of the hypersensitive response in tobacco using functionally non-polar hrpZ deletion mutations, truncated HrpZ fragments, and hrmA mutations; Alfano JR et al.; Pseudomonas syringae pv . syringae, like many plant pathogenic bacteria, secretes a 'harpin' protein that can elicit the hypersensitive response (HR), a defensive cellular suicide, in non-host plants . The harpin-encoding hrpZ gene is located in an operon that also encodes Hrp secretion pathway components and is part of the functional cluster of hrp genes carried on cosmid pHIR11 that enables saprophytic bacteria like Escherichia coli and Pseudomonas fluorescens to elicit the HR in tobacco leaves . We have constructed functionally non-polar hrpZ deletion mutations, revealing that HrpZ is necessary for saprophytic bacteria carrying pHIR11 to elicit a typical HR, whereas it only enhances the elicitation activity of P . s . syringae . Partial deletion mutations revealed that the N-terminal 153 amino acids of HrpZ can enable E . coli MC4100-(pHIR11) to elicit a strong HR . hrpZ subclone products comprising the N-terminal 109 amino acids and C-terminal 216 amino acids, respectively, of the 341 amino acid protein were isolated and found to elicit the HR . P . fluorescens (pHIR11 hrmA::TnphoA) mutants do not elicit the HR, but cell fractionation and immunoblot analysis revealed that they produce and secrete wild-type levels of HrpZ . Therefore, elicitor activity resides in multiple regions of HrpZ, P . syringae produces elicitor(s) in addition to HrpZ, and HrpZ is essential but not sufficient for HR elicitation by saprophytic bacteria carrying pHIR11.

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