Ps. fluorescens

Microbiol Res, 2004, 159(4), 371 - 94
Growth promotion and yield enhancement of peanut (Arachis hypogaea L.) by application of plant growth-promoting rhizobacteria; Dey R et al.; Although plant growth-promoting rhizobacteria (PGPR) have been reported to influence plant growth, yield and nutrient uptake by an array of mechanisms, the specific traits by which PGPR promote plant growth, yield and nutrient uptake were limited to the expression of one or more of the traits expressed at a given environment of plant-microbe interaction . We selected nine different isolates of PGPR from a pool of 233 rhizobacterial isolates obtained from the peanut rhizosphere on the basis of ACC-deaminase activity . The nine isolates were selected, initially, on the basis of germinating seed bioassay in which the root length of the seedling was enhanced significantly over the untreated control . All the nine isolates were identified as Pseudomonas spp . Four of these isolates, viz . PGPR1, PGPR2, PGPR4 and PGPR7 (all fluorescent pseudomonads), were the best in producing siderophore and indole acetic acid (IAA) . In addition to IAA and siderophore-producing attributes, Pseudomonas fluorescens PGPR1 also possessed the characters like tri-calcium phosphate solubilization, ammonification and inhibited Aspergillus niger and A . flavus in vitro . P . fluorescens PGPR2 differed from PGPR1 in the sense that it did not show ammonification . In addition to the traits exhibited by PGPR1, PGPR4 showed strong in vitro inhibition to Sclerotium rolfsii . The performances of these selected plant growth-promoting rhizobacterial isolates were repeatedly evaluated for 3 years in pot and field trials . Seed inoculation of these three isolates, viz . PGPR1, PGPR2 and PGPR4, resulted in a significantly higher pod yield than the control, in pots, during rainy and post-rainy seasons . The contents of nitrogen and phosphorus in soil, shoot and kernel were also enhanced significantly in treatments inoculated with these rhizobacterial isolates in pots during both the seasons . In the field trials, however, there was wide variation in the performance of the PGPR isolates in enhancing the growth and yield of peanut in different years . Plant growth-promoting fluorescent pseudomonad isolates, viz . PGPR1, PGPR2 and PGPR4, significantly enhanced pod yield (23-26%, 24-28% and 18-24%, respectively), haulm yield and nodule dry weight over the control in 3 years . Other attributes like root length, pod number, 100-kernel mass, shelling out-turn and nodule number were also enhanced . Seed bacterization with plant growth-promoting P . fluorescens isolates, viz . PGPR1, PGPR2 and PGPR4, suppressed the soil-borne fungal diseases like collar rot of peanut caused by A . niger and PGPR4 also suppressed stem rot caused by S . rolfsii . Studies on the growth patterns of PGPR isolates utilizing the seed leachate as the sole source of C and N indicated that PGPR4 isolate was the best in utilizing the seed leachate of peanut, cultivar JL24 . Studies on the rhizosphere competence of the PGPR isolates, evaluated on the basis of spontaneous rifampicin resistance, indicated that PGPR7 was the best rhizoplane colonizer and PGPR1 was the best rhizosphere colonizer . Although the presence of growth-promoting traits in vitro does not guarantee that an isolate will be plant growth promoting in nature, results suggested that besides ACC-deaminase activity of the PGPR isolates, expression of one or more of the traits like suppression of phytopathogens, solubilization of tri-calcium phosphate, production of siderophore and/or nodulation promotion might have contributed to the enhancement of growth, yield and nutrient uptake of peanut.

Microbiol Res, 2004, 159(4), 355 - 63
Sequential activation of constitutive and inducible nitric oxide synthase (NOS) in rat cerebellar granule neurons by pseudomonas fluorescens and invasive behaviour of the bacteria; Mezghani-Abdelmoula S et al.; Previous studies have shown that Pseudomonas fluorescens and its lipopolysaccharide (LPS) exert dose-related cytotoxic effects on neurons and glial cells . In the present work, we investigated the time course effect of P . fluorescens MF37 and its LPS on cultured rat cerebellar granule neurons . The kinetics of binding of P . fluorescens to cerebellar granule neurons is rapid and reaches a mean of 3 bacteria/cell after 5 h . As demonstrated by measurement of the concentration of nitrite in the culture medium, P . fluorescens induces a rapid stimulation (3 h) of the nitric oxide synthase (NOS) activity of the cells . In contrast, LPS extracted from P . fluorescens requires a long lag phase (24 h) before observation of an activation of NOS . Measurement of the membrane resting potential of granule neurons showed that within 3 h of incubation there was no difference of effect between the action of P . fluorescens and that of its endotoxin . Two complementary approaches allowed to demonstrate that P . fluorescens MF37 presents a rapid invasive behaviour suggesting a mobilisation of calcium in its early steps of action . The present study reveals that P . fluorescens induces the sequential activation of a constitutive calcium-dependent NOS and that of an inducible NOS activated by LPS . Our results also suggest that in P . fluorescens cytotoxicity and invasion are not mutually exclusive events.

Can J Microbiol, 2004 Oct, 50(10), 811 - 820
Strategy to select and assess antagonistic bacteria for biological control of Rhizoctonia solani Kuhn; Faltin F et al.; A screening strategy was developed to assess the potential of plant-associated bacteria to control diseases caused by Rhizoctonia solani Kuhn . About 434 already characterized antagonistic bacterial strains isolated from diverse plant species and microenvironments were evaluated for biocontrol and plant growth promotion by a hierarchical combination of assays . Analyzing in vitro antagonism towards different Rhizoctonia isolates resulted in a selection of 20 potential biocontrol agents . The strains were characterized by their antagonistic mechanisms in vitro as well as their production of the plant growth hormone indole-3-acetic acid . The plant growth promoting effect by antagonistic bacteria was determined using a microtiter plate assay on the basis of lettuce seedlings . Lettuce and sugar beet as host plant were included in the biocontrol experiments in which the antagonistic effect of 17 bacterial isolates could be confirmed in vivo . Sequencing of the 16S rDNA gene and (or) fatty acid methyl ester gas chromatography was used to identify the antagonistic isolates . Molecular fingerprints of isolates obtained by BOX – polymerase chain reaction were compared to avoid further investigation with genetically very similar strains and to obtain unique molecular fingerprints for quality control and patent licensing . According to our strategy, an assessment scheme was developed and four interesting biological control agents, Pseudomonas reactans B3, Pseudomonas fluorescens B1, Serratia plymuthica B4, and Serratia odorifera B6, were found . While S . plymuthica B4 was the best candidate to biologically control Rhizoctonia in lettuce, P . reactans B3 was the best candidate to suppress the pathogen in sugar beet.

Appl Environ Microbiol, 2005 Jan, 71(1), 542 - 6
Biocidal Activity of Formaldehyde and Nonformaldehyde Biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in Pure and Mixed Suspensions in Synthetic Metalworking Fluid and Saline; Selvaraju SB et al.; The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF . The results indicated that M . immunogenum is more resistant than P . fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol) . Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF . In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline . Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio) . The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.

Res Microbiol, 2005 Jan-Feb, 156(1), 7 - 16
Characterisation of the regulatory RNA RsmB from Pseudomonas aeruginosa PAO1; Burrowes E et al.; In Pseudomonas aeruginosa, the molecular regulation of virulence factors and secondary metabolites is tightly controlled . This control involves several signal-mediated regulatory networks, including the GacS-GacA system and quorum sensing . Recently, the posttranscriptional repressor protein RsmA has been implicated in secondary metabolite production . RsmA is postulated to work in tandem with an as yet unidentified regulatory RNA molecule in a manner analogous to its homologues in other bacteria . Here we have identified a gene encoding an untranslated regulatory RNA (RsmB), located in the rpoS/ fdxA intergenic region of the P . aeruginosa PAO1 genome . Overexpression of rsmB in P . aeruginosa resulted in an increase in N-acyl-homoserine lactone, pyocyanin and elastase production compared with a marked decrease when rsmA was overexpressed . Mutation of rsmB resulted in a decrease in AHL production compared to wild type . We propose that RsmB is the cognate regulatory RNA of RsmA in P . aeruginosa . The global regulator GacA was not absolutely required for rsmB transcription in P . aeruginosa, as is the case in Pseudomonas fluorescens . However, GacA influenced the kinetics of rsmB transcription in that in late stationary phase the gacA mutant showed a substantial reduction in rsmB transcript levels compared to wild type . RsmA also influenced rsmB; in an rsmA mutant, the steady state level of rsmB transcript was reduced and this was due to a decrease in the transcription of rsmB . A balance in the levels of RsmA and RsmB may be an autoregulatory mechanism to ensure that RsmA is tightly controlled, as might be expected for such a potent global repressor.

Peptides, 2005 Feb, 26(2), 277 - 83
Isolation of alliumin, a novel protein with antimicrobial and antiproliferative activities from multiple-cloved garlic bulbs; Xia L et al.; A protein designated alliumin, with a molecular mass of 13kDa and an N-terminal sequence similar to a partial sequence of glucanase, and demonstrating antifungal activity against Mycosphaerella arachidicola, but not against Fusarium oxysporum, was isolated from multiple-cloved garlic (Allium sativum) bulbs . The protein, designated as alliumin, was purified using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Mono S, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75 . Alliumin was unadsorbed on DEAE-cellulose, but was adsorbed on Affi-gel blue gel, CM-cellulose and Mono S . Its antifungal activity was retained after boiling for 1h and also after treatment with trypsin or chymotrypsin (1:1, w/w) for 30min at room temperature . Alliumin was inhibitory to the bacterium Pseudomonas fluorescens and exerted antiproliferative activity toward leukemia L1210 cells . However, it was devoid of ribonuclease activity, protease activity, mitogenic activity toward mouse splenocytes, and antiproliferative activity toward hepatoma Hep G2 cells.

Environ Pollut, 2005 Apr, 134(3), 485 - 92
Biosensor-based diagnostics of contaminated groundwater: assessment and remediation strategy; Bhattacharyya J et al.; Shallow groundwater beneath a former airfield site in southern England has been heavily contaminated with a wide range of chlorinated solvents . The feasibility of using bacterial biosensors to complement chemical analysis and enable cost-effective, and focussed sampling has been assessed as part of a site evaluation programme . Five different biosensors, three metabolic (Vibrio fischeri, Pseudomonas fluorescens 10568 and Escherichia coli HB101) and two catabolic (Pseudomonas putida TVA8 and E . coli DH5alpha), were employed to identify areas where the availability and toxicity of pollutants is of most immediate environmental concern . The biosensors used showed different sensitivities to each other and to the groundwater samples tested . There was generally a good agreement with chemical analyses . The potential efficacy of remediation strategies was explored by coupling sample manipulation to biosensor tests . Manipulation involved sparging and charcoal treatment procedures to simulate remediative engineering solutions . Sparging was sufficient at most locations.

J Agric Food Chem, 2004 Dec 29, 52(26), 8057 - 65
Characterization of Cry34Ab1 and Cry35Ab1 insecticidal crystal proteins expressed in transgenic corn plants and Pseudomonas fluorescens; Gao Y et al.; Cry34Ab1 and Cry35Ab1 proteins, identified from Bacillus thuringiensis strain PS149B1, act together to control corn rootworms . Transgenic corn lines coexpressing the two proteins were developed to protect corn against rootworm damage . Large quantities of the two proteins were needed to conduct studies required for assessing the safety of this transgenic corn crop . Because it was technically infeasible to obtain sufficient quantities of high purity Cry34Ab1 and Cry35Ab1 proteins from the transgenic corn plants, the proteins were produced using a recombinant Pseudomonas fluorescens (Pf) production system . The two proteins from both the transgenic corn and the Pf were purified and characterized . The proteins from each host had the expected molecular mass and were immunoreactive to specific antibodies in enzyme-linked immunosorbent assay and Western blot analysis . Data from N-terminal sequencing, tryptic peptide mass fingerprinting, internal peptide sequencing, and biological activity provided direct evidence that the Cry34Ab1 and Cry35Ab1 proteins produced in Pf and transgenic corn were, respectively, comparable or equivalent molecules . In addition, neither protein had detectable glycosylation regardless of the host.

Environ Biosafety Res, 2004 Apr-Jun, 3(2), 83 - 90
Effects of temperature on detection of plasmid or chromosomally encoded gfp- and lux-labeled Pseudomonas fluorescens in soil; Bunker ST et al.; Pseudomonas fluorescens is a normal inhabitant of the soil rhizosphere . The use of genetically altered strains of P . fluorescens in bioremediation has led to the need for effective monitoring of such cells released into the environment . In this study, we present data on the persistence in soil of P . fluorescens harboring gfp (green fluorescent protein) or lux (bioluminescence) genes . Comparisons were made between strains marked chromosomally and strains carrying these markers on a plasmid . Overall effects of plasmid carriage on culturability were also examined . Sterile soil microcosms were inoculated with washed cells to a final concentration of ca . 10(6) CFU.g(-1) and placed at 5, 23, and 35-37 degrees C . Samples were taken periodically and examined for culturability and viability, using the substrate responsiveness assay . Our results indicated no significant loss of culturability at 5 and 23 degrees C for a period of over one year . In contrast, cells of P . fluorescens incubated at 35-37 degrees C entered the viable but nonculturable state within 7 days . All cells labeled with gfp retained fluorescence regardless of culturability, suggesting that the green fluorescent protein can be of value in monitoring the presence of cells following their release to the environment . Because fluorescence was maintained regardless of the cells' physiological state, this protein may also be an indicator of cell viability.

J Appl Microbiol, 2005, 98(1), 43 - 55
Phenylacetic acid-producing Rhizoctonia solani represses the biosynthesis of nematicidal compounds in vitro and influences biocontrol of Meloidogyne incognita in tomato by Pseudomonas fluorescens strain CHA0 and its GM derivatives; Siddiqui IA et al.; Abstract i.a . siddiqui and s.s . shaukat . 2004.Aims: The aim of the present investigation was to determine the influence of Rhizoctonia solani and its pathogenicity factor on the production of nematicidal agent(s) by Pseudomonas fluorescens strain CHA0 and its GM derivatives in vitro and nematode biocontrol potential by bacterial inoculants in tomato . Methods and Results: One (Rs7) of the nine R . solani isolates from infected tomato roots inhibited seedling emergence and caused root rot in tomato . Thin layer chromatography revealed that culture filtrates of two isolates (Rs3 and Rs7) produced brown spots at Rf-values closely similar to synthetic phenylacetic acid (PAA), a phytotoxic factor . Filtrates from isolate Rs7, amended with the growth medium of P . fluorescens, markedly repressed nematicidal activity and PhlA'-'LacZ reporter gene expression of the bacteria in vitro . On the contrary, isolate Rs4 enhanced nematicidal potential of a 2,4-diacetylphloroglucinol overproducing mutant, CHA0/pME3424, of P . fluorescens strain CHA0 in vitro . Therefore, R . solani isolates Rs4 and Rs7 were tested more rigorously for their potential to influence biocontrol effectiveness of the bacterial agents . Methanol extract of the culture filtrates of PAA-producing isolate Rs7 resulting from medium amended with phenylalanine enhanced fungal repression of the production of nematicidal agents by bacteria, while amendments with zinc or molybdenum eliminated such fungal repression, thereby restoring bacterial potential to cause nematode mortality in vitro . A pot experiment was carried out, 3-week-old tomato seedlings were infested with R . solani isolates Rs4 or Rs7 and/or inoculated with Meloidogyne incognita, the root-knot nematode . The infested soil was treated with aqueous cell suspensions (10(8) CFU) of P . fluorescens strain CHA0 or its GM derivatives or left untreated (as a control) . Observations taken 45 days after nematode inoculation revealed that, irrespective of the bacterial treatments, galling intensity per gram of fresh tomato roots was markedly higher in soil amended with isolate Rs4 than in Rs7-amended soils . Soil amendments with R . solani and the bacterial antagonists resulted in substantial reductions of the number of galls per gram of root . These results are contradictory to those obtained under in vitro conditions where culture filtrates of PAA-positive Rs7 repressed the production of nematicidal compounds . Plants grown in Rs7-amended soils, with or without bacterial inoculants, had lesser shoot and root weights than plants grown in nonamended or Rs4-amended soils . Moreover, amendments with Rs7 substantially retarded root growth and produced necrotic lesions that reduced the number of entry sites for invasion and subsequent infection by nematodes . Populations of P . fluorescens in the tomato rhizosphere were markedly higher in Rs7-amended soils . Conclusions: PAA-producing virulent R . solani drastically affects the potential of P . fluorescens to cause death of M . incognita juveniles in vitro and influences bacterial effectiveness to suppress nematodes in tomato roots . Significance and Impact of the Study: As most agricultural soils are infested with root-infecting fungi, including R . solani, it is likely that some PAA-producing isolates of the fungus may also be isolated from such soils . The inhibitory effect of PAA-producing R . solani on the biosynthesis of nematicidal agent(s) critical in biocontrol may reduce or even eliminate the effectiveness of fluorescent pseudomonads against root-knot nematodes, both in nursery beds and in field conditions . Introduction of bacterial inoculants, for the control of any plant pathogen, should be avoided in soils infested with PAA-producing R . solani . Alternatively, the agents could be applied together with an appropriate quantity of fungicide or chemicals such as zinc to create an environment more favourable for bacterial biocontrol action.

Prikl Biokhim Mikrobiol, 2004 Nov-Dec, 40(6), 654 - 8
{Biodegradation of phenanthrene by Pseudomonas bacteria bearing rhizospheric plasmids in model plant-microbial associations}; Posttranscriptional repression of GacS/GacA-controlled genes by the RNA-binding protein RsmE acting together with RsmA in the biocontrol strain Pseudomonas fluorescens CHA0; Departement de Microbiologie Fondamentale, Batiment de Biologie, Universite de Lausanne, CH-1015 Lausanne Dorigny, Switzerland . Cornelia.Reimmann@unil.ch

In the plant-beneficial soil bacterium Pseudomonas fluorescens CHA0, the production of biocontrol factors (antifungal secondary metabolites and exoenzymes) is controlled at a posttranscriptional level by the GacS/GacA signal transduction pathway involving RNA-binding protein RsmA as a key regulatory element . This protein is assumed to bind to the ribosome-binding site of target mRNAs and to block their translation . RsmA-mediated repression is relieved at the end of exponential growth by two GacS/GacA-controlled regulatory RNAs RsmY and RsmZ, which bind and sequester the RsmA protein . A gene (rsmE) encoding a 64-amino-acid RsmA homolog was identified and characterized in strain CHA0 . Overexpression of rsmE strongly reduced the expression of target genes (hcnA, for a hydrogen cyanide synthase subunit; aprA, for the main exoprotease; and phlA, for a component of 2,4-diacetylphloroglucinol biosynthesis) . Single null mutations in either rsmA or rsmE resulted in a slight increase in the expression of hcnA, aprA, and phlA . By contrast, an rsmA rsmE double mutation led to strongly increased and advanced expression of these target genes and completely suppressed a gacS mutation . Both the RsmE and RsmA levels increased with increasing cell population densities in strain CHA0; however, the amount of RsmA showed less variability during growth . Expression of rsmE was controlled positively by GacA and negatively by RsmA and RsmE . Mobility shift assays demonstrated specific binding of RsmE to RsmY and RsmZ RNAs . The transcription and stability of both regulatory RNAs were strongly reduced in the rsmA rsmE double mutant . In conclusion, RsmA and RsmE together account for maximal repression in the GacS/GacA cascade of strain CHA0.

FEMS Microbiol Lett, 2004 Dec 15, 241(2), 243 - 8
Regulation of the aprX-lipA operon of Pseudomonas fluorescens B52: differential regulation of the proximal and distal genes, encoding protease and lipase, by ompR-envZ; McCarthy CN et al.; The production of lipase and protease from psychrotrophic strains of Pseudomonas fluorescens may result in spoilage of dairy products . The lipase (lipA) and alkaline metalloprotease (aprX) genes of P . fluorescens B52 are regulated by temperature and are located at opposite ends of an operon which contains eight genes and spans 14 kb . In this report, we show that lipase activity in the supernatant of cultures of P . fluorescens strain B52 is also regulated by the homologue of the Escherichia coli EnvZ-OmpR two-component regulatory system . Differences in the regulation of lipase and protease may be related to the proximal and distal locations of aprX and lipA within the operon.

Protein Expr Purif, 2005 Jan, 39(1), 124 - 9
Purification, refolding, and characterization of recombinant Pseudomonas fluorescens lipase; Kim KR et al.; Thermostable Pseudomonas fluorescens SIK W1 lipase (PFL), which is responsible for the spoilage of milk, was overexpressed as inclusion bodies in Escherichia coli . Renaturation of solubilized PFL was achieved by using size-exclusion protein refolding chromatography . The renatured enzyme was purified homogeneously using a combination of gel filtration and ion-exchange FPLC . Its specific activity was found to be enhanced in the presence of Ca(2+) . Secondary structural changes induced by Ca(2+) were monitored by circular dichroism, which demonstrated that the activity increase of PFL in the presence of Ca(2+) is strongly correlated with significant increases in alpha-helix and beta-sheet content . In the presence of Ca(2+), the PFL structure was found resistant to denaturation by guanidine hydrochloride and to enzyme activity loss due to cosolvents like DMSO and trifluoroethanol, suggesting that Ca(2+) plays an important role in inducing conformational changes and consequently in maintaining enzyme structural stability.

Lipids, 2004 Jul, 39(7), 667 - 73
Antibacterial and xanthine oxidase inhibitory cerebrosides from Fusarium sp . IFB-121, an endophytic fungus in Quercus variabilis; Shu RG et al.; Two antibacterial and xanthine oxidase inhibitory cerebrosides, one of which is chemically new, were characterized from the chloroform-methanol (1:1) extract of Fusarium sp . IFB-121, an endophytic fungus in Quercus variabilis . By means of chemical and spectral methods {IR, electrospray ionization MS (ESI-MS), tandem ESI-MS, 1H and 13C NMR, distortionless enhancement by polarization transfer, COSY, heteronuclear multiple-quantum coherence, heteronuclear multiple-bond correlation, and 2-D nuclear Overhauser effect correlation spectroscopy}, the structure of the new metabolite named fusaruside was established as (2S,2'R,3R,3'E,4E,8E,10E)-1-O-beta-D-glucopyranosyl-2-N-(2'-hydroxy-3'-octadecenoyl)-3-hydroxy-9-methyl-4,8,10-sphingatrienine, and the structure of the other was identified as (2S,2'R,3R,3'E,4E,8E)-1-O-beta-D-glucopyranosyl-2-N-(2'-hydroxy-3'-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine . Both new and known cerebrosides, although inactive to Trichophyton rubrum and Candida albicans, showed strong antibacterial activities against Bacillus subtilis, Escherichia coli, and Pseudomonas fluorescens, with their minimum inhibitory concentrations being 3.9, 3.9, and 1.9 microg/mL, and 7.8, 3.9, and 7.8 microg/mL, respectively . Furthermore, both metabolites were inhibitory to xanthine oxidase, with the IC50 value of fusaruside being 43.8 +/- 3.6 microM and the known cerebroside being 55.5 +/- 1.8 microM.

Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2343 - 5 Epub 2004 Dec.
Purification, crystallization and preliminary X-ray crystallographic analysis of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), a crotonase homologue active in phenylpropanoid metabolism; Leonard PM et al.; 4-Hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), also called feruloyl-CoA hydratase-lyase (FCHL), from Pseudomonas fluorescens strain AN103 is an enzyme of the crotonase superfamily that catalyses the one-step conversion of the CoA thioesters of 4-coumaric acid, caffeic acid and ferulic acid to the aromatic aldehydes 4-hydroxybenzaldehyde, protocatechuic aldehyde and vanillin, respectively . The reaction occurs via a hydration followed by a carbon-carbon bond-cleavage reaction . HCHL has been crystallized by the hanging-drop method of vapour diffusion using polyethylene glycol 20 000 Da as the precipitant . The crystals belong to the orthorhombic system, with proposed space group P2(1)2(1)2 and unit-cell parameters a = 154.2, b = 167.5, c = 130.8 A . The V(M) suggests that the asymmetric unit contains four trimers . Single-wavelength data collection has been undertaken and structure determination is under way by molecular replacement using data collected to 1.8 A resolution . Determination of the structure of HCHL will provide insight into the catalytic mechanism of an unusual enzymatic reaction with relevance to the applications of the enzyme in metabolic engineering.

J Biol Chem . 2004 Dec 6; {Epub ahead of print}
Tandemly duplicated acyl carrier proteins which increase polyketide antibiotic production can apparently function either in parallel or in series; Rahman AS et al.; Polyketide biosynthesis involves addition of subunits commonly derived from malonate or methylmalonate to a starter unit such as acetate . Type I polyketide synthases are multifunctional polypeptides that contain one or more modules, each of which normally contains all the enzymatic domains for a single round of extension and modification of the polyketide backbone . Acyl carrier proteins hold the extender unit to which the starter or growing chain is added . Normally there is one ACP for each ketosynthase module . However, there are an increasing number of known examples of tandemly repeated ACP domains, whose function is as yet unknown . For the doublet and triplet ACP domains in the biosynthetic pathway for the antibiotic mupirocin from Pseudomonas fluorescens NCIB10586 we have inactivated ACP domains by in-frame deletion and amino acid substitution of the active site serine . By deletion analysis each individual ACP from a cluster can provide a basic but reduced activity for the pathway . In the doublet cluster, substitution analysis indicates that the pathway may follow two parallel routes, one via each of the ACPs, thus increasing overall pathway flow . In the triplet cluster, substitution in ACP5 blocked the pathway . Thus ACP5 appears to be arranged "in series" to ACP6 and 7 . Thus while both the doublet and triplet clusters increase antibiotic production, the mechanisms by which they do this appear to be different and depend specifically on the biosynthetic stage involved . The function of some ACPs may be determined by their location in the protein rather than absolute enzymic activity.

Am J Gastroenterol, 2004 Dec, 99(12), 2376 - 84
Sero-reactivity to microbial components in Crohn's disease is associated with disease severity and progression, but not NOD2/CARD15 genotype; Arnott ID et al.; BACKGROUND AND AIMS: Antibodies directed against the porin protein C of Escherichia coli (anti-OmpC) and Pseudomonas fluorescens (anti-I2) have recently been described in Crohn's disease (CD) . Those directed against Saccharomyces cerevisiae (ASCA) and the perinuclear component of neutrophils (pANCA) have been more widely studied and may be of diagnostic importance . We aimed to assess the frequency of anti-OmpC, anti-I2, ASCA, and pANCA, in an independent Scottish CD cohort, establish phenotypic associations, and compare with a U.S . cohort . METHODS: One hundred and forty-two well-characterized CD patients (76 females, median age 39 yr (17-88)) were studied . CD was classified by the Vienna classification . Sera were assayed for anti-OmpC, anti-I2, ASCA, and pANCA . Allele specific primers were used for NOD2/CARD15 genotyping . RESULTS: Anti-OmpC, anti-I2, ASCA, and pANCA were present in sera from 37%, 52%, 39%, and 14% of CD patients, respectively . Multivariate analysis demonstrated independent associations of anti-OmpC to be progression of disease type (p= 0.005) and long disease duration (p= 0.002), and those of anti-I2 to be long disease duration (p= 0.002) and the need for surgery (p= 0.033) . ASCA were associated with disease progression (p < 0.001) . When the presence and magnitude of all antibody responses were considered, reactivity to microbial components was associated with long disease duration (p < 0.001), progression of disease type (p < 0.001), penetrating disease (p= 0.008), small bowel disease (p < 0.02), and the need for surgery (p < 0.001) . There was no association of antibody status to NOD2/CARD15 genotype . CONCLUSION: Reactivity to microbial components is associated with severe CD characterized by small bowel involvement, frequent disease progression, longer disease duration, and greater need for intestinal surgery.

J Endod, 2004 Dec, 30(12), 893 - 8
Real-time imaging and quantification of bioluminescent bacteria in root canals in vitro; Sedgley C et al.; The detection of microorganisms in root canals is generally limited to qualitative or semiquantitative methods . We describe a new and nondestructive in vitro method to quantify root-canal bacteria over sequential treatment procedures using real-time imaging in conjunction with the bioluminescent reporter strain Pseudomonas fluorescens 5RL . Induced bacterial photon emission can be monitored by sensitive optical photonic imaging and luminometry, providing images as well as spatial and quantitative measurements . Bioluminescence imaging and luminometry determined that the lower limit of detection of bacteria in root canals occurred between 2 x 10(2) and 2 x 10(3) cells, with high correlation between cell counts and detection devices (r > or = 0.981) . A preliminary study assessed the efficacy of sequential irrigation procedures to remove 5 x 10(6) bacteria from root canals (n = 5; apical size 60) using a 28-gauge, endodontic needle positioned 1 mm from working length; 9.2% +/- 3.1% and 8% +/- 3.6% of bacteria remained after 3 and 6 ml irrigation, respectively (p = 0.03), corresponding to approximately 4 x 10(5) bacteria remaining after 6 ml . This method can be used to study the efficacy of sequential endodontic treatment procedures in removing bacteria from root canals.

Mol Plant Microbe Interact, 2004 Nov, 17(11), 1201 - 11
Defense responses of Fusarium oxysporum to 2,4-diacetylphloroglucinol, a broad-spectrum antibiotic produced by Pseudomonas fluorescens; Schouten A et al.; A collection of 76 plant-pathogenic and 41 saprophytic Fusarium oxysporum strains was screened for sensitivity to 2,4-diacetylphloroglucinol (2,4-DAPG), a broad-spectrum antibiotic produced by multiple strains of antagonistic Pseudomonas fluorescens . Approximately 17% of the F . oxysporum strains were relatively tolerant to high 2,4-DAPG concentrations . Tolerance to 2,4-DAPG did not correlate with the geographic origin of the strains, formae speciales, intergenic spacer (IGS) group, or fusaric acid production levels . Biochemical analysis showed that 18 of 20 tolerant F . oxysporum strains were capable of metabolizing 2,4-DAPG . For two tolerant strains, analysis by mass spectrometry indicated that deacetylation of 2,4-DAPG to the less fungitoxic derivatives monoacetylphloroglucinol and phloroglucinol is among the initial mechanisms of 2,4-DAPG degradation . Production of fusaric acid, a known inhibitor of 2,4-DAPG biosynthesis in P . fluorescens, differed considerably among both 2,4-DAPG-sensitive and -tolerant F . oxysporum strains, indicating that fusaric acid production may be as important for 2,4-DAPG-sensitive as for -tolerant F . oxysporum strains . Whether 2,4-DAPG triggers fusaric acid production was studied for six F . oxysporum strains; 2,4-DAPG had no significant effect on fusaric acid production in four strains . In two strains, however, sublethal concentrations of 2,4-DAPG either enhanced or significantly decreased fusaric acid production . The implications of 2,4-DAPG degradation, the distribution of this trait within F . oxysporum and other plant-pathogenic fungi, and the consequences for the efficacy of biological control are discussed.

FEMS Microbiol Lett, 2004 Dec 1, 241(1), 13 - 20
Pseudomonas fluorescens infection by bacteriophage PhiS1: the influence of temperature, host growth phase and media; Sillankorva S et al.; The influence of host growth temperature, phase and media, together with the effect of infection temperature on bacteriophage PhiS1 infection of Pseudomonas fluorescens were examined . The rates of cell lysis and phage release were determined and showed that the efficacy of phage infection was optimal with host cells grown and infected at 26 degrees C . The host physiological state also affected these rates . Infection was dependent on the presence of cell wall proteins with molecular weights of 17.5+/-1 and 99+/-5 kDa.

J Food Prot, 2004 Nov, 67(11), 2560 - 4
Determination of thermal inactivation kinetics of microorganisms with a continuous microflow apparatus; Loss CR et al.; Use of a continuous microflow submerged microcoil (CSMC) apparatus was compared with the capillary tube (CT) method for measuring the thermal inactivation kinetics of Pseudomonas fluorescens at 61 degrees C for 3 to 29 s . Inocula were continuously pumped through a microbore (< or = 0.0762 cm inside diameter) thin-walled stainless steel capillary tube submerged in a heated oil bath . The heating time was set by changing the flow rate, tube dimensions, or both . With the use of microthermo-couples, the time for the inocula to reach within 1 degree C of the set temperature was <3 s, and shorter than that with capillary tubes or vials . Inactivation curves (61 degrees C) for P . fluorescens prepared by the CSMC method were not different from curves prepared by the CT method, as determined by analysis of variance (P > 0.05) . Inactivation of Bacillus cereus spores (105 degrees C) and native microflora found in raw milk (72 degrees C) over heating times of 3 to 42 s were determined by CSMC . CSMC can measure thermal inactivation kinetics of microorganisms efficiently and simply at high temperatures and in short times . Survivors can be enumerated in 1-ml volumes of heat-treated samples, making it useful for determining inactivation kinetics of low numbers of microorganisms, such as those found in high-quality raw milk . Inactivation kinetics were generally more accurately described by the Weibull function (R2 > or = 0.97) than the linear kinetic model.

J Food Prot, 2004 Nov, 67(11), 2397 - 402
Spread of marker bacteria from the hides of cattle in a simulated livestock market and at an abattoir; Collis VJ et al.; The spread of microbial contamination on the hides of beef was investigated at two stages in the meat chain: (i) in a simulated livestock market ("the market") using 33 animals, and (ii) in the unloading-to-skinning area of a commercial abattoir using 18 animals . At both stages, harmless bacterial markers (nalidixic acid-resistant Escherichia coli K-12; rifampicin- and nalidixic acid-resistant Pseudomonas fluorescens; and a tetracycline-resistant E . coli) were inoculated on the hides of a small number of selected animals, and their transfer to other animals and the environment was examined . At the market, the initial prevalence of animals positive for the hide markers (9.1% in each phase) introduced in the presale pen, sale ring, and postsale pen changed to 39.4, 15.1, and 54.5%, respectively, by the end of the market process . In addition, widespread contamination of the market environment with the hide markers was observed . At the abattoir, the initial prevalence of animals positive for the hide marker (11.1%) inoculated at unloading increased to 100% (hide before skinning) and 88.8% (skinned carcass) . In addition, another marker inoculated on environmental surfaces in lairage pens, races, and stunning box was detected on 83.3% (hide before skinning) and 88.8% (skinned carcass) . These results, although obtained with a relatively small number of animals, demonstrate that both the livestock market process and the unloading-to-skinning process at abattoirs can facilitate the extensive spread of microbial contamination on hides not just within, but also between, batches of animals.

Mol Plant Microbe Interact, 2004 Nov, 17(11), 1185 - 91
Role of chemotaxis toward fusaric acid in colonization of hyphae of Fusarium oxysporum f . sp . radicis-lycopersici by Pseudomonas fluorescens WCS365; de Weert S et al.; Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings . The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f . sp . radicis-lycopersici . Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria . Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization . To that end, an in vitro assay was developed to study hyphae colonization by bacteria . The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together . Cells of WCS365 show a chemotactic response toward the spent growth medium of F . oxysporum f . sp . radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not . Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant . Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants . A positive correlation was found between chemo-attractant activity and fusaric acid level . No chemotactic response was observed toward the low fusaric acid-producer FO242 . Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process . The possible function of hyphae colonization for the bacterium is discussed.

J Biol Chem . 2004 Nov 17; {Epub ahead of print}
Aluminum triggers decreased aconitase activity via Fe-S cluster disruption and the overexpression of isocitrate dehydrogenase and isocitrate lyase: A metabolic network mediating cellular survival; Middaugh J et al.; Although aluminum (Al) is known to be toxic to most organisms, its precise biochemical interactions are not fully understood . In the present study, we demonstrate that Al promotes the inhibition of aconitase activity (Acn) via the perturbation of the Fe-S cluster in Pseudomonas fluorescens . Despite the significant decrease in citrate isomerization activity cellular survival is assured by the overexpression of isocitrate lyase (ICL) and isocitrate dehydrogenase-NADP+ (IDH) . 13C NMR spectroscopic studies, Blue Native (BN) PAGE and Western blot analyses indicate that while the decrease in Acn activity is concomitant with the increase of Al in the culture, the amount of Acn expressed is not sensitive to the concentration of the trivalent metal . A 6-fold decrease in Acn activity and no discernable change in protein content in Al-stressed cultures are observed . The addition of Fe(NH4)2(SO4)2 in a reducing environment leads to significant recovery in Acn activity . This enzymatic activity reverts back to normal levels when Al-stressed cells are transferred to either control or iron-supplemented medium . The overexpression of the two isocitrate metabolizing enzymes ICL and IDH-NADP+ appears to mitigate the deficit in Acn activity . The levels of these enzymes are dependent on the Al-content of the culture and appear to be under transcriptional control . Hence, the regulation of the enzymes involved in the homeostasis of isocitrate constitutes a pivotal component of the global metabolic strategy that ensures the survival of this organism in an Al-citrate environment.

J Appl Microbiol, 2004, 97(6), 1192 - 200
The role of host organism, transcriptional switches and reporter mechanisms in the performance of Hg-induced biosensors; Harkins M et al.; AIMS: The purpose of this study was to comprehensively compare the response of nine biosensors capable of being induced by Hg . Induction by Hg was based upon the insertion of merR, merB, zntA and zntR promoter genes . LuxCDABE or lucFF reporter genes expressed luminescence, and host organisms were Escherichia coli, Vibrio anguillarum and Pseudomonas fluorescens . The role of transcriptional switches, reporter mechanism and host organism was to be investigated . METHODS AND RESULTS: All biosensors were subjected to the same assay conditions . Sensors had their own individual growth characteristics and response to the doses of Hg tested . Maximum bioluminescence response was induced by concentrations of Hg between 2.5 nm and 5 microM . E . coli pRB28 was found to detect levels of Hg as low as 1.6 nm and yet was capable of operating in a concentration range of up to 12.5 microM . CONCLUSIONS: The response of the sensors demonstrated their suitability for analysis under environmentally relevant concentrations . The sensitivity of the sensors, the optimum range and the expediency of the assay could not be related to a single sensor trait . It may be concluded that biosensor performance is dependent on more than one of the single factors studied . SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that comparative testing of sensors is an important step in evaluating the relevance and performance of biosensors prior to routine environmental application.

Proc Natl Acad Sci U S A, 2004 Nov 23, 101(47), 16431 - 6 Epub 2004 Nov 23.
Structure and function of the phenazine biosynthetic protein PhzF from Pseudomonas fluorescens; Blankenfeldt W et al.; Phenazines produced by Pseudomonas and Streptomyces spp . are heterocyclic nitrogen-containing metabolites with antibiotic, antitumor, and antiparasitic activity . The antibiotic properties of pyocyanin, produced by Pseudomonas aeruginosa, were recognized in the 1890s, although this blue phenazine is now known to be a virulence factor in human disease . Despite their biological significance, the biosynthesis of phenazines is not fully understood . Here we present structural and functional studies of PhzF, an enzyme essential for phenazine synthesis in Pseudomonas spp . PhzF shares topology with diaminopimelate epimerase DapF but lacks the same catalytic residues . The structure of PhzF in complex with its substrate, trans-2,3-dihydro-3-hydroxyanthranilic acid, suggests that it is an isomerase using the conserved glutamate E45 to abstract a proton from C3 of the substrate . The proton is returned to C1 of the substrate after rearrangement of the double-bond system, yielding an enol that converts to the corresponding ketone . PhzF is a dimer that may be bifunctional, providing a shielded cavity for ketone dimerization via double Schiff-base formation to produce the phenazine scaffold . Our proposed mechanism is supported by mass and NMR spectroscopy . The results are discussed in the context of related structures and protein sequences of unknown biochemical function.

Microbiology, 2004 Nov, 150(Pt 11), 3889 - 97
Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization; Capdevila S et al.; The ability of plant-associated micro-organisms to colonize and compete in the rhizosphere is specially relevant for the biotechnological application of micro-organisms as inoculants . Pseudomonads are one of the best root colonizers and they are widely used in plant-pathogen biocontrol and in soil bioremediation . This study analyses the motility mechanism of the well-known biocontrol strain Pseudomonas fluorescens F113 . A 6.5 kb region involved in the flagellar filament synthesis, containing the fliC, flaG, fliD, fliS, fliT and fleQ genes and part of the fleS gene, was sequenced and mutants in this region were made . Several non-motile mutants affected in the fliC, fliS and fleQ genes, and a fliT mutant with reduced motility properties, were obtained . These mutants were completely displaced from the root tip when competing with the wild-type F113 strain, indicating that the wild-type motility properties are necessary for competitive root colonization . A mutant affected in the flaG gene had longer flagella, but the same motility and colonization properties as the wild-type . However, in rich medium or in the absence of iron limitation, it showed a higher motility, suggesting the possibility of improving competitive root colonization by manipulating the motility processes.

Mikrobiologiia, 2004 Jul-Aug, 73(4), 504 - 10
{A comparative analysis of the ice nucleation activity of pseudomonad cells and lipopolysaccharides}; Elemental and redox analysis of single bacterial cells by x-ray microbeam analysis; Environmental Research Division and Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439-4843, USA . kemner@anl.gov

High-energy x-ray fluorescence measurements were used to make elemental maps and qualitative chemical analyses of individual Pseudomonas fluorescens strain NCIMB 11764 cells . Marked differences between planktonic and adhered cells were seen in the morphology, elemental composition, and sensitivity to Cr(VI) of hydrated cells at spatial scales of 150 nm . This technology can be applied to natural geomicrobiological systems.

Nature, 2004 Oct 21, 431(7011), 984 - 8
Ecological constraints on diversification in a model adaptive radiation; Kassen R et al.; Taxonomic diversification commonly occurs through adaptive radiation, the rapid evolution of a single lineage into a range of genotypes or species each adapted to a different ecological niche . Radiation size (measured as the number of new types) varies widely between phylogenetically distinct taxa and between replicate radiations within a single taxon where the ecological opportunities available seem to be identical . Here we show how variation in energy input (productivity) and environmental disturbance combine to determine the extent of diversification in a single radiating lineage of Pseudomonas fluorescens adapting to laboratory conditions . Diversity peaked at intermediate rates of both productivity and disturbance and declined towards the extremes in a manner reminiscent of well-known ecological patterns . The mechanism responsible for the decrease in diversity arises from pleiotropic fitness costs associated with niche specialization, the effects of which are modulated by gradients of productivity and disturbance . Our results indicate that ecological gradients may constrain the size of adaptive radiations, even in the presence of the strong diversifying selection associated with ecological opportunity, by decoupling evolutionary diversification from ecological coexistence.

J Bacteriol, 2004 Nov, 186(21), 7411 - 9
Use of in vivo expression technology to identify genes important in growth and survival of Pseudomonas fluorescens Pf0-1 in soil: discovery of expressed sequences with novel genetic organization; Silby MW et al.; Studies were undertaken to determine the genetic needs for the survival of Pseudomonas fluorescens Pf0-1, a gram-negative soil bacterium potentially important for biocontrol and bioremediation, in soil . In vivo expression technology (IVET) identified 22 genes with elevated expression in soil relative to laboratory media . Soil-induced sequences included genes with probable functions of nutrient acquisition and use, and of gene regulation . Ten sequences, lacking similarity to known genes, overlapped divergent known genes, revealing a novel genetic organization at those soil-induced loci . Mutations in three soil-induced genes led to impaired early growth in soil but had no impact on growth in laboratory media . Thus, IVET studies have identified sequences important for soil growth and have revealed a gene organization that was undetected by traditional laboratory approaches.

Pol J Microbiol, 2004, 53(2), 101 - 10
An attempt to protect winter wheat against Gaeumannomyces graminis var . tritici by the use of rhizobacteria Pseudomonas fluorescens and Bacillus mycoides; Czaban J et al.; Pseudomonas fluorescens strains III107 and II21 and Bacillus mycoides strains JC192 and K184, stimulating growth of winter wheat, were chosen for the studies . The bacterial strains inhibited on agar nutrient medium the growth of Gaeumannomyces graminis var . tritici (Ggt)--the pathogenic fungus causing take-all on wheat . Both strains of pseudomonads synthesized relatively high amounts of Fe3+ chelators . The strains of bacilli were characterized by the very fast spreading on agar media . Furthermore, strain II21 was highly cyanogenic, and strain JC192 highly chitinolytic . Bacterization of winter wheat seeds (especially with strains III107 and JC192) significantly reduced the percentage of the plants infested with the pathogen in the 28 day glasshouse pot experiment . In the plot experiment, the winter wheat seeds were inoculated with a mixture of strains III107, II21 and JC192 . Due to the bacterization the yield of wheat grain and straw was higher in comparison to the series with Ggt alone by 122% and 75%, respectively, but it amounted only to 45% and 43% of the control series not contaminated with Ggt . The decrease of percentage of wheat ears with weight less than 500 mg from 61% in Ggt-series to 25% in Ggt-bacterized-series, and especially the decrease of percentage of wheat ears with weight less than 200 mg from 43% to 14% additionally indicate the partial protection of the winter wheat against Ggt by the rhizobacteria . In the experimental series not contaminated with Ggt the percentage of these wheat ears fractions did not exceed 3% and 0.5%, respectively.

Appl Environ Microbiol, 2004 Oct, 70(10), 6342 - 6
Isolation of lightning-competent soil bacteria; Ceremonie H et al.; Artificial transformation is typically performed in the laboratory by using either a chemical (CaCl(2)) or an electrical (electroporation) method . However, laboratory-scale lightning has been shown recently to electrotransform Escherichia coli strain DH10B in soil . In this paper, we report on the isolation of two "lightning-competent" soil bacteria after direct electroporation of the Nycodenz bacterial ring extracted from prairie soil in the presence of the pBHCRec plasmid (Tc(r), Sp(r), Sm(r)) . The electrotransformability of the isolated bacteria was measured both in vitro (by electroporation cuvette) and in situ (by lightning in soil microcosm) and then compared to those of E . coli DH10B and Pseudomonas fluorescens C7R12 . The electrotransformation frequencies measured reached 10(-3) to 10(-4) by electroporation and 10(-4) to 10(-5) by simulated lightning, while no transformation was observed in the absence of electrical current . Two of the isolated lightning-competent soil bacteria were identified as Pseudomonas sp . strains.

FEMS Microbiol Lett, 2004 Oct 1, 239(1), 17 - 23
Intragenomic heterogeneity of the 16S rRNA-23S rRNA internal transcribed spacer among Pseudomonas syringae and Pseudomonas fluorescens strains; Milyutina IA et al.; The 16S-23S rRNA internal transcribed spacer regions (ITS1) from 14 strains of Pseudomonas syringae and P . fluorescens were sequenced . ITS1 exhibited significant sequence variability among different operons within a single genome . From 1 to 4 types of ITS1 were found in individual genomes of the P . syringae and P . fluorescens strains . A total of eight ITS1 types were identified among strains studied . The ITS1 nucleotide sequences consisted of conserved blocks including, among others, a stem-forming region of box B, tRNAIle and tRNAAla genes and several variable blocks . The differences in the variable regions were mostly due to insertions and/or deletions of nucleotide blocks . The intragenomic heterogeneity of ITS1 was brought about by different combinations of variable blocks, which possibly have resulted from recombination and horizontal transfer.

Biochemistry, 2004 Oct 5, 43(39), 12427 - 35
Structure and function of the phenazine biosynthesis protein PhzF from Pseudomonas fluorescens 2-79; Parsons JF et al.; Phenazines, including pyocyanin and iodonin, are biologically active compounds that are believed to confer producing organisms with a competitive growth advantage, and also are thought to be virulence factors in certain diseases including cystic fibrosis . The basic, tricyclic phenazine ring system is synthesized in a series of poorly characterized steps by enzymes encoded in a seven-gene cistron in Pseudomonas and other organisms . Despite the biological importance of these compounds, and our understanding of their mode of action, the biochemistry and mechanisms of phenazine biosynthesis are not well resolved . Here we report the 1.8 A crystal structure of PhzF, a key enzyme in phenazine biosynthesis, solved by molecular replacement . PhzF is structurally similar to the lysine biosynthetic enzyme diaminopimelate epimerase, sharing an unusual fold consisting of two nearly identical domains with the active site located in an occluded cleft between the domains . Unlike diaminopimelate epimerase, PhzF is a dimer in solution . The two apparently independent active sites open toward opposite sides of the dimer and are occupied by sulfate ions in the structure . In vitro experiments using a mixture of purified PhzF, -A, -B, and -G confirm that phenazine-1-carboxylic acid (PCA) is readily produced from trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) without aid of other cellular factors . PhzA, -B, and -G have no activity toward DHHA . However, in the presence of PhzF, individually or in combinations, they accelerate the formation of PCA from DHHA and therefore appear to function after the action of PhzF . Surprisingly, PhzF is itself capable of producing PCA, albeit slowly, from DHHA . These observations suggest that PhzF catalyzes the initial step in the conversion of DHHA to PCA, probably via a rearrangement reaction yielding the more reactive 3-oxo analogue of DHHA, and that subsequent steps can occur spontaneously . A hypothetical model for how DHHA binds to the PhzF active site suggests that Glu45 and Asp208 could act as general acid-base catalysts in a rearrangement reaction . Given that four reactions lie between DHHA and PCA, ketone formation, ring formation, decarboxylation, and oxidation, we hypothesize that the similar PhzA and -B proteins catalyze ring formation and thus may be more than noncatalytic accessory proteins . PhzG is almost certainly an oxidase and is predicted to catalyze the final oxidation/aromatization reaction.

Can J Microbiol, 2004 Jul, 50(7), 475 - 81
Colonization and persistence of a plant growth-promoting bacterium Pseudomonas fluorescens strain CS85, on roots of cotton seedlings; Wang C et al.; Pseudomonas fluorescens CS85, which was previously isolated from the rhizosphere of cotton seedlings, acts as both a plant growth-promoting bacterium and a biocontrol agent against cotton pathogens, including Rhizoctonia solani, Colletotrichum gossypii, Fusarium oxysporum f sp . vasinfectum, and Verticillium dahliae . Strain CS85 was labeled separately with luxAB and gusA . The labeled strains were stably maintained and had high levels of expression of the marker genes, luxAB and gusA, after successive transfers on nonselective medium, long-term preservation, and after recovery from soil . The labeled strains displayed similar biocontrol characteristics (e.g., antibiosis, effects of growth-promotion and disease-control) to the original strain . The labeled strains colonized all surfaces of the young plant root zones, such as roots hairs and lateral roots, although the distribution of the labeled strains on the root surfaces was not uniform . Moreover, the population densities of the labeled strains on the root surface were stably maintained at high levels during the first 2 weeks of plant growth in the native soil, so that about 10(7)-10(8) CFU/g root were detected, then decreased gradually . Nevertheless, approximately 10(6) CFU/g root of the labeled strains were observed on the root surfaces 35 d after planting.

Arch Microbiol, 2004 Oct, 182(2-3), 147 - 56 Epub 2004 Aug 31.
Microorganisms degrading chlorobenzene via a meta-cleavage pathway harbor highly similar chlorocatechol 2,3-dioxygenase-encoding gene clusters; Gobel M et al.; Pseudomonas putida GJ31 harbors a degradative pathway for chlorobenzene via meta-cleavage of 3-chlorocatechol . Pseudomonads using this route for chlorobenzene degradation, which was previously thought to be generally unproductive, were isolated from various contaminated environments of distant locations . The new isolates, Pseudomonas fluorescens SK1 (DSM16274), Pseudomonas veronii 16-6A (DSM16273), Pseudomonas sp . strain MG61 (DSM16272), harbor a chlorocatechol 2,3-dioxygenase (CbzE) . The cbzE-like genes were cloned, sequenced, and expressed from the isolates and a mixed culture . The chlorocatechol 2,3-dioxygenases shared 97% identical amino acids with CbzE from strain GJ31, forming a distinct family of catechol 2,3-dioxygenases . The chlorocatechol 2,3-dioxygenase, purified from chlorobenzene-grown cells of strain SK1, showed an identical N-terminal sequence with the amino acid sequence deduced from cloned cbzE . In all investigated chlorobenzene-degrading strains, cbzT-like genes encoding ferredoxins are located upstream of cbzE . The sequence data indicate that the ferredoxins are identical (one amino acid difference in CbzT of strain 16-6A compared to the others) . In addition, the structure of the operon downstream of cbzE is identical in strains GJ31, 16-6A, and SK1 with genes cbzX (unknown function) and the known part of cbzG (2-hydroxymuconic semialdehyde dehydrogenase) and share 100% nucleotide sequence identity with the entire downstream region . The current study suggests that meta-cleavage of 3-chlorocatechol is not an atypical pathway for the degradation of chlorobenzene.

J Ethnopharmacol, 2004 Oct, 94(2-3), 301 - 5
Evaluation of antibacterial properties of some medicinal plants used in Iran; Bonjar S; Forty-five species of 29 plant families used in the traditional medicine by Iranian people, showed antibacterial activities against one or more of the bacterial species: Bacillus cereus, Bacillus pumilus, Bordetella bronchiseptica, Escherichia coli, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia marcescens, Staphylococcus aureus and Staphylococcus epidermidis . No plant showed activity against Serratia marcescens; Bordetella bronchiseptica being the most susceptible species . All extracts showed the same activity 18 months later.

Mikrobiologiia, 2004 May-Jun, 73(3), 312 - 9
{Structure and properties of the lipopolysaccharide of Pseudomonas fluorescens IMV 2366 (biovar III)}; Veremeichenko SN et al.; The lipopolysaccharide (LPS) preparation isolated from the bacterial mass of Pseudomonas fluorescens IMV 2366 (biovar III) by Westphal's method and purified by repeated ultracentrifugation was characterized by the presence of the S- and R-forms of molecules . The following structural portions of the LPS molecule were obtained in the individual state and characterized: lipid A, core oligosaccharide, and O-specific polysaccharide . The main components of the lipid A hydrophobic moiety were 3-hydoxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, and hexadecanoic fatty acids . Glucosamine, phosphoethanolamine, and phosphorus were identified as the components of the lipid A hydrophilic moiety . Rhamnose, glucose, galactose, glucosamine, galactosamine, alanine, phosphoethanolamine, phosphorus, 2-keto-3-desoxyoctulosonic acid (KDO), as well as 2-amino-2,6-didesoxygalactose (FucN) and 3-amino-3,6-didesoxyglucose (Qui3N), were revealed in the composition of the core oligosaccharide fractions . O-specific polysaccharide chains were established to be composed of repeating trisaccharide units consisting of residues of L-rhamnose (L-Rha), 2-acetamido-2,6-didesoxy-D-galactose (D-FucNAc), and 3-acylamido-3,6-didesoxy-D-glucose (D-Qui3NAcyl), where Acyl = 3-hydroxy-2,3-dimethyl-5-hydroxyprolyl . Neither double immunodiffusion in agar not the immunoenzyme assay revealed serological relations between the strain studied and the P . fluorescens strains studied earlier.

Mol Plant Microbe Interact, 2004 Aug, 17(8), 895 - 908
The transcriptome of rhizobacteria-induced systemic resistance in arabidopsis; Verhagen BW et al.; Plants develop an enhanced defensive capacity against a broad spectrum of plant pathogens after colonization of the roots by selected strains of nonpathogenic, fluorescent Pseudomonas spp . In Arabidopsis thaliana, this rhizobacteria-induced systemic resistance (ISR) functions independently of salicylic acid but requires responsiveness to the plant hormones jasmonic acid and ethylene . In contrast to pathogen-induced systemic acquired resistance, rhizobacteria-mediated ISR is not associated with changes in the expression of genes encoding pathogenesis-related proteins . To identify ISR-related genes, we surveyed the transcriptional response of over 8,000 Arabidopsis genes during rhizobacteria-mediated ISR . Locally in the roots, ISR-inducing Pseudomonas fluorescens WCS417r bacteria elicited a substantial change in the expression of 97 genes . However, systemically in the leaves, none of the approximately 8,000 genes tested showed a consistent change in expression in response to effective colonization of the roots by WCS417r, indicating that the onset of ISR in the leaves is not associated with detectable changes in gene expression . After challenge inoculation of WCS417r-induced plants with the bacterial leaf pathogen P . syringae pv . tomato DC3000, 81 genes showed an augmented expression pattern in ISR-expressing leaves, suggesting that these genes were primed to respond faster or more strongly upon pathogen attack . The majority of the primed genes was predicted to be regulated by jasmonic acid or ethylene signaling . Priming of pathogen-induced genes allows the plant to react more effectively to the invader encountered, which might explain the broad-spectrum action of rhizobacteria-mediated ISR.

Acta Crystallogr D Biol Crystallogr, 1996 Mar, 52(Pt 2), 393 - 401
Refined Crystal Structure of the Catalytic Domain of Xylanase A from Pseudomonas fluorescens at 1.8 A Resolution; Harris GW; The three-dimensional structure of native xylanase A from Pseudomonas flouorescens subspecies cellulosa has been refined at 1.8 A resolution . The space group is P2(1)2(1)2(1) with four molecules in the asymmetric unit . The final model has an R factor of 0.166 for 103 749 reflections with the four molecules refined independently . The tertiary structure consists of an eightfold beta/alpha-barrel, the so-called TIM-barrel fold . The active site is in an open cleft at the carboxy-terminal end of the beta/alpha-barrel, and the active-site residues are a pair of glutamates, Glu127 on strand 4 and Glu246 on strand 7 . Both these catalytic glutamate residues are found on beta-bulges . An atypically long loop after strand 7 is stabilized by calcium . Unusual features include a non-proline cis-peptide residue Ala80 which is found on a beta-bulge at the end of beta-strand 3 . The three beta-bulge type distortions occurring on beta-strands 3, 4 and 7 are functionally significant as they serve to orient important active-site residues . The active-site residues are further held in place by an extensive hydrogen-bonding network of active-site residues in the catalytic site of xylanase A . A chain of well ordered water molecules occupies the substrate-binding cleft, some or all of which are expelled on binding of the substrate.

Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 175 - 8
Optimization of the biodegradation of naphthalene by a microorganism isolated from petroleum contaminated soil; Martin A et al.; Response surface methodology (RSM) was used to optimise the process parameters to obtain maximum biodegradation of naphthalene . The factorial design employed, a face-centred cube, helped in identifying the combined best parameter conditions . Naphthalene-containing medium was inoculated with Pseudomonas fluorescens and incubated at different levels of agitation, pH, and temperature, and analyzed by Gas Chromatography-Mass Spectroscopy . As a result of the optimisation process, the process parameters found for maximum degradation of naphthalene by P . fluorescens were an agitation speed of 186 rpm, a pH of 7.3, and a temperature of 22.8 degrees C.

Appl Environ Microbiol, 2004 Aug, 70(8), 4666 - 71
Effect of nematodes on rhizosphere colonization by seed-applied bacteria; Knox OG et al.; There is much interest in the use of seed-applied bacteria for biocontrol and biofertilization, and several commercial products are available . However, many attempts to use this strategy fail because the seed-applied bacteria do not colonize the rhizosphere . Mechanisms of rhizosphere colonization may involve active bacterial movement or passive transport by percolating water or plant roots . Transport by other soil biota is likely to occur, but this area has not been well studied . We hypothesized that interactions with soil nematodes may enhance colonization . To test this hypothesis, a series of microcosm experiments was carried out using two contrasting soils maintained under well-defined physical conditions where transport by mass water flow could not occur . Seed-applied Pseudomonas fluorescens SBW25 was capable of rhizosphere colonization at matric potentials of -10 and -40 kPa in soil without nematodes, but colonization levels were substantially increased by the presence of nematodes . Our results suggest that nematodes can have an important role in rhizosphere colonization by bacteria in soil.

Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1438 - 40 Epub 2004 Jul 21.
Crystallization and X-ray diffraction of a halogenating enzyme, tryptophan 7-halogenase, from Pseudomonas fluorescens; Dong C et al.; Chlorination of natural products is often required for their biological activity; notable examples include vancomycin, the last-ditch antibiotic . It is now known that many chlorinated natural products are made not by haloperoxidases, but by FADH2-dependent halogenases . The mechanism of the flavin-containing enzymes is obscure and there are no structural data . Here, crystals of PrnA (tryptophan 7-halogenase), an enzyme that regioselectively chlorinates tryptophan, cocrystallized with tryptophan and FAD are reported . The crystals belong to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 67.8, c = 276.9 A . A data set to 1.8 A with 93% completeness and an Rmerge of 7.1% has been collected from a single flash-cooled crystal . A method for incorporating selenomethionine in a Pseudomonas fluorescens expression system also is reported.

Colloids Surf B Biointerfaces, 2004 May 15, 35(2), 143 - 7
Impact of lipopolysaccharide coating on clay particle wettability; Chen G et al.; Impact of lipopolysaccharide coating on kaolinite and Na-montmorillonite wettability was investigated . Kaolinite had greater diiodomethane contact angles, smaller water and formamide contact angles than Na-montmorillonite . After lipopolysaccharide coating, diiodomethane and formamide contact angles decreased, while water contact angles increased for both kaolinite and Na-montmorillonite . The decrease and increase in liquid contact angles after lipopolysaccharide coating were most pronounced for lipopolysaccharide extracted from Pseudomonas aeruginosa, followed by Pseudomonas fluorescens and Echerichia coli . Clay particle wettability was determined by particle surface thermodynamic properties . Both kaolinite and Na-montmorillonite exhibited a monopolar surface and the monopolarity decreased after lipopolysaccharide coating, indicating an increase in hydration or surface wetness . The origins of interactions of clay particles with water molecules were discussed and related to clay particle water wettability.

Colloids Surf B Biointerfaces, 2004 Jul 15, 36(2), 75 - 80
Adhesion of Pseudomonas fluorescens on magnetic surfaces; Yeo SH et al.; The adhesion of Pseudomonas fluorescens (ATCC 700830) to perpendicularly polarized magnetic surfaces was recently discovered . The findings have found that the magnetic free surfaces from different magnetic polarities have different profound effects on the P . fluorescens bacterial adhesion to its surfaces . These phenomena can be explained by the surface magnetic effect, which was found to affect the surface free energy . An in situ experiment, by contrast microscopy and under static conditions, was conducted to determine the influence of magnetic surfaces, that are polarized under different external magnetizing field strengths, on bacterial adhesion . The effect of different magnetic polarities on the surface free energy has also been investigated.

Microbiology, 2004 Jul, 150(Pt 7), 2443 - 50
The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance; Abbas A et al.; 2,4-Diacetylphloroglucinol (PHL) is the primary determinant of the biological control activity of Pseudomonas fluorescens F113 . The operon phlACBD encodes enzymes responsible for PHL biosynthesis from intermediate metabolites . The phlE gene, which is located downstream of the phlACBD operon, encodes a putative permease suggested to be a member of the major facilitator superfamily with 12 transmembrane segments . PhlE has been suggested to function in PHL export . Here the sequencing of the phlE gene from P . fluorescens F113 and the construction of a phlE null mutant, F113-D3, is reported . It is shown that F113-D3 produced less PHL than F113 . The ratio of cell-associated to free PHL was not significantly different between the strains, suggesting the existence of alternative transporters for PHL . The phlE mutant was, however, significantly more sensitive to high concentrations of added PHL, implicating PhlE in PHL resistance . Furthermore, the phlE mutant was more susceptible to osmotic, oxidative and heat-shock stresses . Osmotic stress induced rapid degradation of free PHL by the bacteria . Based on these results, we propose that the role of phlE in general stress tolerance is to export toxic intermediates of PHL degradation from the cells.

FEMS Microbiol Lett, 2004 Jul 15, 236(2), 349 - 57
Fluorescence resonance energy transfer (FRET) based molecular detection of a genetically modified PCB degrader in soil; Hogan J et al.; Genetic analysis of the location of a mini-Tn5 promoted insertion of the LB400 bph operon in the rhizosphere coloniser Pseudomonas fluorescens F113rifPCB, allowed the development of a specific PCR detection system based on the unique DNA sequence at this insertion site . Real time PCR using both SYBR green chemistry and Fluorescence Resonance Energy Transfer probes allowed the precise identification of the recombinant strain and its quantitative detection in soil microcosms over a (bacteria/g) range of five orders of magnitude . This new assay can detect the genetically modified microorganism from soil in less than 90 min and at levels below the detection limits of standard PCR or cultivable counts on selective media.

FEMS Microbiol Lett, 2004 Jul 15, 236(2), 257 - 60
Development of PCR assay to identify Pseudomonas fluorescens and its biotype; Scarpellini M et al.; The paper provides a simple protocol that uses the polymerase chain reaction to amplify a specific portion of the 16S gene, allowing the recognition of Pseudomonas fluorescens from other group I Pseudomonas . The amplified DNA patterns of 16S rRNA and ITS1, from the restriction fragment length polymorphisms VspI, HaeIII and TaqI digestion, produced band patterns that distinguished the biotypes of Ps . fluorescens . In addition to distinguishing the biotypes C and 3 we used a phenotypical method for levan production.

Res Microbiol, 2004 Jul-Aug, 155(6), 467 - 74
Bacterial deposition in porous medium as impacted by solution chemistry; Chen G et al.; Bacterial transport in porous medium was investigated by means of column experiments using typical rod-shaped bacteria of Escherichia coli and Pseudomonas fluorescens . Mobility of E . coli and P . fluorescens in silica gel decreased with increasing ionic strength of the solution . In the presence of nonionic surfactants, the mobility of E . coli and P . fluorescens increased, and this was more pronounced at lower than at higher ionic strength . Bacterial transport in the porous medium was described by the equilibrium-kinetic two-region model and bacterial deposition was assumed to occur in the kinetic adsorption region only . Quantified bacterial deposition from bacterial column breakthrough curves was related to electrostatic and Lifshitz-van der Waals interactions between bacterial cells and medium surfaces . It was found that electrostatic interactions played a more important role than Lifshitz-van der Waals interactions in determining bacterial deposition in the porous medium, and were actually the barrier for bacteria to attach to the porous medium.

Bioresour Technol, 2004 Nov, 95(2), 223 - 7
Effects of plant growth promoting bacteria and composed organic fertilizers on the reproduction of Meloidogyne incognita and tomato growth; Siddiqui ZA; Glasshouse experiments were conducted to assess the influence of Pseudomonas fluorescens, Azotobacter chroococcum, Azospirillum brasilense and composted organic fertilizers (cow dung, horse dung, goat dung and poultry manure) alone and in combination on the multiplication of Meloidogyne incognita and growth of tomato . P . fluorescens was better at improving tomato growth and reducing galling and nematode multiplication than A . chroococcum or A . brasilense . Among composted organic fertilizers, poultry manure resulted in less galling and nematode multiplication than occurred with goat dung . However, composted goat dung was better in reducing nematode multiplication and improving plant growth than horse dung . Cow dung was the composted organic fertilizer least effective in reducing galling and nematode multiplication . Poultry manure with P . fluorescens was the best combination for the management of M . incognita on tomato but improved management of M . incognita can also be obtained if goat dung is used with P . fluorescens or poultry manure with A . chroococcum.

Biochemistry (Mosc), 2004 Jun, 69(6), 674 - 7
Cloning and sequencing of the gene of tryptophan-7-halogenase from Pseudomonas fluorescens strain CHA0; Burd VN et al.; The gene of tryptophan-7-halogenase from the Pseudomonas fluorescens strain CHA0, a producer of the halogenated antibiotic pyrrolnitrin, has been cloned and sequenced.

J Chromatogr A, 2004 Jun 4, 1038(1-2), 267 - 73
Use of immobilized lipases for lipase purification via specific lipase-lipase interactions; Palomo JM et al.; Lipase from Pseudomonas fluorescens (PFL), an enzyme with a great tendency to yield bimolecular aggregates, was immobilized via multipoint covalent attachment on glyoxyl-agarose in the presence of Triton X-100 . This strategy permitted to obtain the enzyme with the active center oriented towards the reaction medium . This immobilized enzyme presents the capacity of specifically adsorbing PFL molecules, that can be easily desorbed by the use of detergents . More interesting, the enzyme was also able to adsorb other lipases . That is, the lipase from Bacillus thermocatenulatus (BTL2) cloned in Escherichia coli was selectively adsorbed on this immobilized enzyme, enabling a very simple purification strategy . Similar results were achieved with some other lipases (those from Rhizomucor miehei (RML), Rhizopus oryzae (ROL), and Humicola Lanuginosa (HLL)) . In all cases, the enzyme could be easily desorbed by incubation with Triton X-100 . The matrix could be used several cycles without any detrimental effect on the adsorption capacity.

Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1289 - 91 Epub 2004 Jun 22.
Crystallization and preliminary analysis of xenobiotic reductase B from Pseudomonas fluorescens I-C; Orville AM et al.; Single crystals have been obtained of xenobiotic reductase B (XenB), a flavoenzyme isolated and cloned from Pseudomonas fluorescens I-C . The enzyme catalyzes the NADPH-dependent elimination of nitrite from nitroglycerin with an approximately fivefold kinetic preference for the middle nitro group, primarily yielding 1,3-dinitroglycerol . X-ray diffraction data sets have been collected from native crystals to 2.3 A resolution . The space group is P4(1)2(1)2, with unit-cell parameters a = b = 140, c = 95.6 A . The asymmetric unit is likely to contain at least two XenB molecules (V(M) = 3.1 A(3) Da(-1), 60% solvent) and a molecular-replacement solution has been determined in order to solve the structure.

Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1237 - 43 Epub 2004 Jun 22.
Structure of an aryl esterase from Pseudomonas fluorescens; Cheeseman JD et al.; The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 A by X-ray diffraction and shows a characteristic alpha/beta-hydrolase fold . In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity . PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s . deviation in 271 C(alpha) atoms between PFE and its five closest structural neighbors averaging 0.8 A . PFE has far less similarity (r.m.s . deviation in 218 C(alpha) atoms of 5.0 A) to P . fluorescens carboxyl esterase . PFE favors activated esters with small acyl groups, such as phenyl acetate . The X-ray structure of PFE reveals a significantly occluded active site . In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.

Mikrobiol Z, 2004 Mar-Apr, 66(2), 3 - 10
{Taxonomic contribution and antagonistic properties of antarctic fluorescent bacteria of Pseudomonas genus}; Kotsofliak OI et al.; Fifty (50) strains of Pseudomonas genus have been isolated from the samples of soil, moss and bottom sediments taken on the Galindes, Piterman and Deception Islands (Ukrainian Antarctic station Akademik Vernadsky) . Characteristic of the above strains by 84 phenotype features and identification by means of the computer program permitted them to be referred to the species Pseudomonas fluorescens (biovar V.), P . putida (biovars A and B), P . veronii . The belonging of the strain 23 to the species P . veronii has been confirmed by the results of sequence of 16S rRNA . Being grown at low temperatures (4 degrees C) the strains of the above species are characterized by high antagonistic activity as to Gram-positive, Gram-negative bacteria, yeast and microscopic fungi . Cultivation of antagonists at 26 degrees C led to the sharp decrease or complete disappearance of the antagonistic effect.

Mycorrhiza, 2004 Jul, 14(3), 185 - 92 Epub 2003 Aug 06.
Impact of two fluorescent pseudomonads and an arbuscular mycorrhizal fungus on tomato plant growth, root architecture and P acquisition; Gamalero E et al.; The ability of fluorescent pseudomonads and arbuscular mycorrhizal fungi (AMF) to promote plant growth is well documented but knowledge of the impact of pseudomonad-mycorrhiza mixed inocula on root architecture is scanty . In the present work, growth and root architecture of tomato plants (Lycopersicon esculentum Mill . cv . Guadalete), inoculated or not with Pseudomonas fluorescens 92rk and P190r and/or the AMF Glomus mosseae BEG12, were evaluated by measuring shoot and root fresh weight and by analysing morphometric parameters of the root system . The influence of the microorganisms on phosphorus (P) acquisition was assayed as total P accumulated in leaves of plants inoculated or not with the three microorganisms . The two bacterial strains and the AMF, alone or in combination, promoted plant growth . P . fluorescens 92rk and G . mosseae BEG12 when co-inoculated had a synergistic effect on root fresh weight . Moreover, co-inoculation of the three microorganisms synergistically increased plant growth compared with singly inoculated plants . Both the fluorescent pseudomonads and the myco-symbiont, depending on the inoculum combination, strongly affected root architecture . P . fluorescens 92rk increased mycorrhizal colonization, suggesting that this strain is a mycorrhization helper bacterium . Finally, the bacterial strains and the AMF, alone or in combination, improved plant mineral nutrition by increasing leaf P content . These results support the potential use of fluorescent pseudomonads and AMF as mixed inoculants for tomato and suggest that improved tomato growth could be related to the increase in P acquisition.

Lett Appl Microbiol, 2004, 39(1), 74 - 83
Differential impact of some Aspergillus species on Meloidogyne javanica biocontrol by Pseudomonas fluorescens strain CHA0; Siddiqui IA et al.; AIMS: The aim was to determine the influence of some Aspergillus species on the production of nematicidal agent(s) in vitro and biocontrol of Meloidogyne javanica in tomato by Pseudomonas fluorescens strains CHA0 and CHA0/pME3424 . METHODS AND RESULTS: Six species of Aspergillus, isolated from the rhizosphere of certain crops, produced a variety of secondary metabolites in vitro . Culture filtrate (CF) obtained from Ps . fluorescens strain CHA0 and its2,4-diacetylphloroglucinol overproducing mutant CHA0/pME3424 grown in King's B liquid medium caused significant mortality of M . javanica juveniles in vitro . Bacterial growth medium amended with CF of A . niger enhanced nematicidal and beta-galactosidase activities of fluorescent pseudomonads while A . quadrilineatus repressed such activities . Methanol or ethyl acetate extracts of the CF of A . niger markedly optimized bacterial efficacy to cause nematode deaths while hexane extract of the fungus had no influence on the nematicidal activity of the bacterial strains . A . niger applied alone or in conjunction with the bacterial inoculants inhibited root-knot nematode galling in tomato . On the other hand, A . quadrilineatus used alone or together with CHA0 did not inhibit nematode galling but when used in combination with strain CHA0/pME3424 did reduce galling intensity . CONCLUSIONS: Aspergillus niger enhances the production of nematicidal compounds by Ps . fluorescensin vitro and improves biocontrol potential of the bacterial inoculants in tomato while A . quadrilineatus reduces bacterial performance to suppress root-knot nematodes . SIGNIFICANCE AND IMPACT OF THE STUDY: Rhizosphere harbours a variety of micro-organisms including bacteria, fungi and viruses . Aspergillus species are ubiquitous in most agricultural soils and generally produce a variety of secondary metabolites . Such metabolites synthesized by Aspergillus species may influence the production of nematicidal agents and subsequent biocontrol performance of the bacterial inoculants against plant-parasitic nematodes . This fact needs to be taken into consideration when using biocontrol strains in an agriculture system.

Electrophoresis, 2004 Jun, 25(10-11), 1536 - 42
Rapid analysis of antimicrobial metabolites monoacetylphloroglucinol and 2,4-diacetylphloroglucinol using capillary zone electrophoresis; Guihen E et al.; A rapid capillary electrophoretic (CE) method was developed for the determination of phloroglucinol compounds, monoacetylphloroglucinol (MAPG) and 2,4-diacetylphloroglucinol (DAPG), in microbial supernatants of Pseudomonas fluorescens F113 over a 24-h growth cycle . Prior to electrophoretic separation, solid-phase extraction of supernatant samples on octadecylsilica for the purpose of sample cleanup is recommended . The optimum electrophoretic conditions were found to be 25 mM sodium tetraborate running buffer at pH 9.3, temperature at 25 degrees C with an applied voltage of 25 kV . The capillary was an Agilent fused-silica capillary of total length 33 cm x 50 microm inner diameter, 375 microm outer diameter, with effective length 24.5 cm . While MAPG and DAPG were monitored at selected wavelengths in the range of 214-320 nm, analysis at 214 nm was used and a CE separation time of less than 2 min was achieved . A partial method validation study was performed in accordance with European Agency for Evaluation of Medicinal Products (EMEA) guidelines . The method displayed linearity over the investigated range of 10-200 microg/mL, with limits of detection of 1.2 microg/mL for MAPG and 1.3 microg/mL for DAPG.

Appl Environ Microbiol, 2004 Jun, 70(6), 3558 - 65
Modeling the rate of attachment of Listeria monocytogenes, Pantoea agglomerans, and Pseudomonas fluorescens to, and the probability of their detachment from, potato tissue at 10 degrees C; Garrood MJ et al.; The rate of attachment of bacteria to, and their subsequent detachment from, the cut surface of raw potato tissue was measured and modeled by using mathematical approaches that allowed detailed objective comparisons of adhesion processes under different conditions . Attachment was rapid and reached equilibrium after contact for 60 min . A new method to measure the probability of detachment was developed and modeled, revealing that the probability of detachment for Pseudomonas fluorescens remained unchanged for contact times between less than 5 s and 60 min . Listeria monocytogenes, however, was more easily removed initially, with the probability of detachment decreasing over the first 2 min of contact but remaining constant and equivalent to that for Pseudomonas fluorescens thereafter . For all of the bacteria tested, the number of bacteria attached after 2 min of contact was proportional to the inoculum concentration raised to the power of 0.79.

Biochim Biophys Acta, 2004 Jun 11, 1672(3), 131 - 4
Cytoplasmic membrane polarization in Gram-positive and Gram-negative bacteria grown in the absence and presence of tetracycline; Vincent M et al.; The ability of numerous diverse compounds and ions to cross the bacterial cytoplasmic membrane by diffusion and active transport is highly dependent on cytoplasmic membrane fluidity, which can be measured using fluorescent probes to estimate membrane polarization values . However, membrane polarization data are lacking for most bacterial species . The cytoplasmic membrane polarization values for Arthrobacter sp . ATCC 21908, Bacillus cereus NRC 3045, Pseudomonas fluorescens R2F, Pseudomonas putida NRC 2986 and Escherichia coli C600 bacterial cells were spectrofluorometrically measured over a temperature range from 10 to 50 degrees C, and in the absence and presence of 1 microg/ml tetracycline, using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to obtain new information on their membrane fluidity . At an assay temperature of 10 degrees C, E . coli cells grown in the absence of tetracycline exhibited the highest cytoplasmic membrane polarization value (least fluid membrane) of 0.446, followed by values of 0.392, 0.371, 0.344 and 0.293, respectively, for B . cereus, Arthrobacter sp., P . fluorescens and P . putida . At an assay temperature of 30 degrees C, the polarization values ranged from 0.357 to 0.288 for cells grown in the absence of tetracycline, regardless of the species . B . cereus grown in the presence of 1 microg/ml tetracycline had lower polarization values than when grown in the absence of this antibiotic at all assay temperatures . Regardless of the absence or presence of 1 microg/ml tetracycline in the growth medium, all bacterial species generally exhibited a more fluid membrane as the assay temperature increased from 10 to 50 degrees C . To our knowledge, these are some of the first cytoplasmic membrane polarization values reported for these Gram-negative and Gram-positive bacteria over a broad temperature range and also for cells grown in the presence of tetracycline.

Biochem Biophys Res Commun, 2004 Jun 25, 319(2), 671 - 6
Cell-free protein synthesis using cell extract of Pseudomonas fluorescens and CspA promoter; Nakashima N et al.; We have modified the cell-free coupled transcription/translation system of bacteria . The cell-free extract of Pseudomonas fluorescens was used for translation instead of Escherichia coli . In addition, transcription of the target gene was regulated by CspA promoter with endogenous RNA polymerase instead of by T7 promoter with exogenous T7 RNA polymerase . We could increase the yields of soluble proteins using different combinations of the S30 extract and the promoter and different temperatures for protein synthesis . Increasing the variety of synthesis systems allows production of large quantities of soluble proteins . In order to carry out efficient cell-free protein synthesis, versatile pCop-plasmids carrying CspA promoter were constructed and these plasmids were applicable to expression of recombinant proteins in E . coli cells.

Rev Argent Microbiol, 2004 Jan-Mar, 36(1), 6 - 15
{Influence of salinity and temperature on fatty acid composition of Pseudomonas fluorescens GNP-OHP-3 membrane}; Pucci GN et al.; The bacteria respond to environmental changes modifying their composition . One of the most important modifications is the variation on fatty acid composition of cellular membranes to maintain the homeoviscosity . The action of temperature, hydrostatic pressure and solvents on Pseudomonas putida has been thoroughly studied . In this paper, the combined action of the temperature and salinity on fatty acid composition of cellular membranes of Pseudomonas fluorescens GNP-OHP-3, a bacterial strain isolated from a petroleum contaminated habitat, was studied . The modifications in the fatty acid composition of Pseudomonas fluorescens GNP-OHP-3 membrane were similar to those described for other members of Pseudomonas: an increase in saturated fatty acids and a decrease in unsaturated fatty acids were observed with the increase of the temperature . Variations of main fatty acids were in general erratic in the range of assayed saline concentrations . The variation of cyclopropane fatty acids could be expressed with mathematic equations that allowed to predict their percentage in relation to sodium chloride concentration.

Biotechnol Lett, 2004 Apr, 26(7), 549 - 57
A study into the anti-microbial properties of an amino functionalised polymer using multi-parameter flow cytometry; Hewitt CJ et al.; Fluorescent staining techniques were used to study the anti-microbial properties of aqueous suspensions of a novel, water insoluble amino functionalised polymer on three micro-organisms Pseudomonas fluorescens, Staphylococcus epidermidis and Saccharomyses cerevisiae . The mechanism of action was similar for each organism in that, after various contact times with the polymer, a progressive change in individual cell physiological state was measured using multi-parameter flow cytometry . The microbiocidal activity of this polymer may be similar to that of substances referred to as polycationic, amphipathic compounds (peptides, peptide derivatives and other polyamines).

Microbiol Res, 2004, 159(1), 73 - 81
Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen; Nagarajkumar M et al.; Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus . Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R . solani in vitro . Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P . fluorescens strains was evaluated . The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2 . A significant relationship between the antagonistic potential of P . fluorescens against R . solani and its level of beta-1,3-glucanase, SA and HCN was observed.

Acta Crystallogr D Biol Crystallogr, 2004 Jun, 60(Pt 6), 1129 - 31 Epub 2004 May 21.
Overexpression, purification and crystallization of PhzA, the first enzyme of the phenazine biosynthesis pathway of Pseudomonas fluorescens 2-79; Ahuja EG et al.; Phenazines are broad-spectrum antibiotic metabolites produced by organisms such as Pseudomonas and Streptomyces . Phenazines have been shown to enhance microbial competitiveness and the pathogenic potential of the organisms that synthesize them . PhzA (163 residues, approximate molecular weight 18.7 kDa) is the product of the first of seven genes of the operon responsible for phenazine biosynthesis in P . fluorescens 2-79 . This enzyme is thought to catalyse one of the final steps in the formation of phenazine-1-carboxylic acid, the end product of phenazine biosynthesis in P . fluorescens 2-79 . Here, the purification and crystallization of recombinant PhzA are reported . Crystals diffracting to 2.1 angstroms were obtained using 1.6 M magnesium sulfate and 2-morpholinoethanesulfonic acid monohydrate (MES) buffer pH 5.2-5.6 . Crystals of both native and seleno-L-methionine-labelled protein belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 66.8, b = 75.3, c = 84.5 angstroms . The asymmetric unit contains one dimer of PhzA .

Bioorg Med Chem, 2004 Jun 15, 12(12), 3333 - 8
Two novel antibiotics, Sch 419558 and Sch 419559, produced by Pseudomonas fluorescens: effect on activity by overexpression of RpoE; Yang SW et al.; Two new secondary metabolites designated as Sch 419558 (1) and Sch 419559 (2), were isolated from the fermentation broth of Pseudomonas fluorescens . Structure elucidation of 1 and 2 was accomplished by spectroscopic data analyses including MS and NMR experiments . Both compounds were identified as lipopeptides containing valine and threonine linked with 1-amino-1-hydroxy-heptadec-9-en-2-one or 1-amino-1-hydroxy-pentadecan-2-one carbon chains, respectively . Characterization of the amino acids was further confirmed by amino acid analysis . Compounds 1 and 2 exhibited antibacterial activity against a sensitized E . coli strain with minimum inhibitory concentration of 0.3 and 0.6 microg/mL, respectively . Overexpression of RpoE in the E . coli strain increased the MIC over 60-fold for compounds 1 and 2.

Biochemistry, 2004 Jun 1, 43(21), 6370 - 7
Structure of the ferrous form of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis in complex with the therapeutic herbicide, NTBC; Brownlee JM et al.; Di- and triketone inhibitors of (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) are both effective herbicides and therapeutics . The inhibitory activity is used to halt the production of lipophilic redox cofactors in plants and also in humans to prevent accumulation of toxic metabolic byproducts that arise from specific inborn defects of tyrosine catabolism . The three-dimensional structure of the Fe(II) form of HPPD from Streptomyces avermitilis in complex with the inhibitor 2-{2-nitro-4-(triflouromethyl)benzoyl}-1,3-cyclohexanedione (NTBC) has been determined at a resolution of 2.5 A . NTBC coordinates to the active site metal ion, located at the bottom of a wide solvent-accessible cavity in the C-terminal domain of the protein . The iron is liganded in a predominantly five-coordinate, distorted square-pyramidal arrangement in which Glu349, His187, and His270 are protein-derived ligands and two other ligands are from the 5' and 7' oxygens of NTBC . There is a low-occupancy water molecule in the sixth coordination site in one of the protomers . The distance to His270 is unusually long at 2.5 A, and its orientation is somewhat distorted from ideal ligand geometry to within 2.8 A of the inhibitor nitro group . In contrast to the tetrameric quartenary structure observed for HPPD from other bacterial sources, the asymmetric unit is composed of two weakly associated protomers with a buried surface area of 1266 A(2) and a total of 12 hydrogen-bonding and no electrostatic interactions . The overall tertiary structure is similar to that of HPPD from Pseudomonas fluorescens (Serre et al., (1999) Structure 7, 977-988), although the position of the C-terminal alpha-helix is dramatically shifted . This C-terminal alpha-helix provides Phe364, which in combination with Phe336 sandwiches the phenyl ring of the bound NTBC; no other significant hydrogen-bonding or charge-pairing interactions are observed . Moreover, the structure reveals that, with the exception of Val189, NTBC makes contacts to only fully conserved amino acids . The combination of bidentate metal-ion coordination and pi-stacked aromatic rings is suggestive of a binding mode for the substrate and/or a transition state, which may be the origin of the exceedingly high affinity these inhibitors have for HPPD.

Antimicrob Agents Chemother, 2004 Jun, 48(6), 2334 - 6
IMP-1 and a novel metallo-beta-lactamase, VIM-6, in fluorescent pseudomonads isolated in Singapore; Koh TH et al.; Four carbapenem-resistant Pseudomonas spp . were isolated from patients in Singapore . One Pseudomonas putida isolate contained a bla(IMP-1) identical to that first described in Japan . The sequence of a variant bla(IMP-1) in Pseudomonas fluorescens contained four silent mutations compared with the original sequence . The remaining P . putida isolates contained bla(VIM-6), a novel VIM gene variant.

Commun Agric Appl Biol Sci, 2003, 68(4 Pt B), 543 - 7
Biological control of Fusarium oxysporum, the causal agent of onion wilt by antagonistic bacteria; Sharifi Tehrani A et al.; Fusarium wilt caused by Fusarium oxysporum Sch . is one of the most important diseases of onion in Iran . Application of chemicals especially as soil drench, increased cost of onion production and may be dangerous for environment . One of the effective techniques to suppress soil-born diseases in biological control with antagonistic rhizobacteria . Experiment were carried out with 120 bacterial isolates that were collected from onion rhizosphere . Six highly effective isolates were selected from these antagonists for subsequent studies . These strains were used to investigate their biocontrol traits in vitro and their ability to suppress the onion wilt in vivo (soil and seed treatments) . According to the biochemical, physiological and morphological test, the isolates 22, 38, 46 and 52 were identified as Bacillus spp . The isolates 16 and 48 were identified as Pseudomonas fluorescens . The isolates of Bacillus spp . produced volatile metabolites that inhibited mycelia growth of Fusarium oxysporum . In soil treatment, the isolates 22 and 52 with 56% and 51% had the highest effect in reducing the Fusarium wilt of onion . The mixture of two isolates reduced 60% the disease . In seed treatment the isolate 22 with 41% had the greatest effect on reducing the onion Fusarium wilt.

Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 8072 - 7 Epub 2004 May 18.
The evolution of a pleiotropic fitness tradeoff in Pseudomonas fluorescens; MacLean RC et al.; The evolution of ecological specialization is expected to carry a cost, due to either antagonistic pleiotropy or mutation accumulation . In general, it has been difficult to distinguish between these two possibilities . Here, we demonstrate that the experimental evolution of niche-specialist genotypes of the bacterium Pseudomonas fluorescens that colonize the air-broth interface of spatially structured microcosms is accompanied by pleiotropic fitness costs in terms of reduced carbon catabolism . Prolonged selection in spatially structured microcosms caused the cost of specialization to decline without loss of the benefits associated with specialization . The decline in the cost of specialization can be explained by either compensatory adaptation within specialist lineages or clonal competition among specialist lineages . These results provide a possible explanation of conflicting accounts for the cost of specialization.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 713 - 9
Psychrophilic pseudomonads from Antarctica: Pseudomonas antarctica sp . nov., Pseudomonas meridiana sp . nov . and Pseudomonas proteolytica sp . nov; Reddy GS et al.; Thirty-one bacteria that belonged to the genus Pseudomonas were isolated from cyanobacterial mat samples that were collected from various water bodies in Antarctica . All 31 isolates were psychrophilic; they could be divided into three groups, based on their protein profiles . Representative strains of each of the three groups, namely CMS 35(T), CMS 38(T) and CMS 64(T), were studied in detail . Based on 16S rRNA gene sequence analysis, it was established that the strains were related closely to the Pseudomonas fluorescens group . Phenotypic and chemotaxonomic characteristics further confirmed their affiliation to this group . The three strains could also be differentiated from each other and the closely related species Pseudomonas orientalis, Pseudomonas brenneri and Pseudomonas migulae, based on phenotypic and chemotaxonomic characteristics and the level of DNA-DNA hybridization . Therefore, it is proposed that strains CMS 35(T) (=MTCC 4992(T)=DSM 15318(T)), CMS 38(T) (=MTCC 4993(T)=DSM 15319(T)) and CMS 64(T) (=MTCC 4994(T)=DSM 15321(T)) should be assigned to novel species of the genus Pseudomonas as Pseudomonas antarctica sp . nov., Pseudomonas meridiana sp . nov . and Pseudomonas proteolytica sp . nov., respectively.

J Bacteriol, 2004 May, 186(10), 3153 - 9
Generation of enhanced competitive root-tip-colonizing Pseudomonas bacteria through accelerated evolution; de Weert S et al.; A recently published procedure to enrich for efficient competitive root tip colonizers (I . Kuiper, G . V . Bloemberg, and B . J . J . Lugtenberg, Mol . Plant-Microbe Interact . 14:1197-1205) after bacterization of seeds was applied to isolate efficient competitive root tip colonizers for both the dicotyledenous plant tomato and the monocotyledenous plant grass from a random Tn5luxAB mutant bank of the good root colonizer Pseudomonas fluorescens WCS365 . Unexpectedly, the best-colonizing mutant, strain PCL1286, showed a strongly enhanced competitive root-tip-colonizing phenotype . Sequence analyses of the Tn5luxAB flanking regions showed that the transposon had inserted in a mutY homolog . This gene is involved in the repair of A . G mismatches caused by spontaneous oxidation of guanine . We hypothesized that, since the mutant is defective in repairing its mismatches, its cells harbor an increased number of mutations and therefore can adapt faster to the environment of the root system . To test this hypothesis, we constructed another mutY mutant and analyzed its competitive root tip colonization behavior prior to and after enrichment . As a control, a nonmutated wild type was subjected to the enrichment procedure . The results of these analyses showed (i) that the enrichment procedure did not alter the colonization ability of the wild type, (ii) that the new mutY mutant was strongly impaired in its colonization ability, but (iii) that after three enrichment cycles it colonized significantly better than its wild type . Therefore it is concluded that both the mutY mutation and the selection procedure are required to obtain an enhanced root-tip-colonizing mutant.

Environ Health Perspect, 2004 May, 112(6), 659 - 65
Synergistic interaction in simultaneous exposure to Streptomyces californicus and Stachybotrys chartarum; Huttunen K et al.; The microbial exposure associated with health complaints in moldy houses consists of a heterogeneous group of components, including both living and dead bacteria, fungi, and their metabolites and active compounds . However, little is known about the interactions between different microbes and their metabolites, although the cytotoxicity and inflammatory potential of certain individual microbes have been reported . In this study, we investigated the inflammatory responses of mouse RAW264.7 macrophages after exposure to six indoor air microbes (Aspergillus versicolor, Penicillium spinulosum, Stachybotrys chartarum, Bacillus cereus, Mycobacterium terrae, and Pseudomonas fluorescens) alone and together with the actinomycete Streptomyces californicus . The production of nitric oxide, levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), and cytotoxicity were measured . The coexposure to Sta . chartarum and Str . californicus caused a synergistic increase in the production of IL-6 but not other cytokines . In further experiments, the metabolites from Sta . chartarum or from closely related fungi (atranones B and E, satratoxin G, trichodermin, 7-alpha-hydroxytrichodermol, staplabin, and SMTP-7) and the known fungal toxins sterigmatocystin, citrinin, and ochratoxin A were each tested with Str . californicus . The testing revealed a synergistic response in TNF-alpha and IL-6 production after coexposure to Str . californicus with both trichodermin and 7-alpha-hydroxytrichodermol . Finally, the synergistic inflammatory response caused by Str . californicus and trichodermin together was studied by analyzing for the presence of nuclear factor-kappa-B (NF-kappa-B) in nuclear extracts of the exposed cells . The exposure to Str . californicus induced the binding of NF-kappa-B proteins to the NF-kappa-B consensus sequence as well as to the natural NF-kappa-B site of the IL-6 promoter . Adding trichodermin to the exposure did not increase the DNA binding.

Biochem Biophys Res Commun, 2004 May 14, 317(4), 1189 - 94
Overexpression of isocitrate lyase is an important strategy in the survival of Pseudomonas fluorescens exposed to aluminum; Hamel R et al.; Isocitrate lyase, ICL (EC 4.1.3.1), an enzyme that cleaves isocitrate into succinate, and glyoxylate appears to play a pivotal role in the detoxification of aluminum (Al) in Pseudomonas fluorescens . Here, we present evidence that the 4-fold increase in ICL activity observed in Al-stressed cells is due to the overexpression of this enzyme . Blue-Native-PAGE, Western blotting, and spectrophotometric experiments revealed that ICL is optimally expressed at 35 h of growth in Al-stressed cells . However, following the immobilization of Al, at 60 h of growth, the level of the enzyme decreases markedly . This enzyme that exists as a homotetramer with a molecular mass of approximately 133 kDa appears to be transcriptionally regulated . The overexpression of ICL may be a specific response to Al-stress as P . fluorescens grown in the presence of such metals as Ga3+, Pb2+, and Ca2+ does not undergo any significant increase in ICL activity . Thus, these findings support the notion that the overexpression of ICL plays a pivotal role in the survival and in the increased oxalogenesis observed in Al-stressed P . fluorescens.

Environ Pollut, 1993, 82(1), 33 - 7
Indium detoxification in Pseudomonas fluorescens; Anderson S et al.; The interaction between indium, a non-essential toxic element, and a soil bacterium was studied . Although the presence of 0.5 mm indium complexed to citrate, the sole source of carbon, had an inhibitory influence on growth rate and cellular yield, Pseudomonas fluorescens circumvented the toxicity of the trivalent metal via its insolubilization as a phosphorus residue . The inclusion of 20 microm iron (III) arrested the negative impact of indium and no diminution of cellular yield was recorded . In this instance indium homeostasis was also attained by elaboration of an extracellular phosphorus-containing deposit . Electrophoretic analyses of the cytoplasmic extracts revealed several dissimilar patterns . Notably, two polypeptides with apparent molecular masses of 57 kDa and 18 kDa were induced in the metal-stressed bacteria . An increment in extracellular carbohydrates in metal-supplemented media was observed . No citrate was detected in the spent fluid at the cessation of cellular bilization may have potential application in metal pollution management.

J Environ Qual, 2004 Mar-Apr, 33(2), 505 - 12
Time and moisture effects on total and bioavailable copper in soil water extracts; Tom-Petersen A et al.; Environmental risk assessment of heavy metals in soil frequently involves testing of freshly spiked soils kept under stable humidity conditions, but it has been questioned whether these assessments are representative of the field situation . Furthermore, the poor correspondence that is often found between total metal content and metal toxicity calls for integrated chemical and biological analysis . The aim of this work was to determine time- and moisture-dependent changes in total water-extractable Cu as well as bioavailable Cu in soil water extracts . Measurements of total water-extractable copper ({Cu}tot) were performed using furnace atomic absorption spectrometry . An in vitro assay employing a Cu-specific Pseudomonas fluorescens reporter strain was used to estimate Cu that was biologically available to the reporter strain . We refer to this copper fraction as "bioavailable," {Cu}bio . We found a time-dependent decrease in {Cu}tot and {Cu}bio during incubation for up to 220 d at field capacity . Hence the {Cu}bio was reduced to between 32 and 40% of the initial values . Furthermore, the {Cu}bio to {Cu}tot ratio correlated positively with the amount of added Cu and tended to increase with time . The moisture content of the soil was important for Cu retention . Dry soil had higher {Cu}tot concentrations than humid soil, but the {Cu}bio to {Cu}tot, ratio was lower in the dry soil . Alternating drying and wetting did not lead to a more rapid Cu retention than observed under constant humid conditions . Our observations underline the need for considering both time and moisture effects when interpreting short-term toxicity studies and when making predictions concerning possible long-term effects of Cu in the soil environment.

Environ Sci Technol, 2004 Mar 15, 38(6), 1740 - 5
Effect of root-derived substrates on the expression of nah-lux genes in Pseudomonas fluorescens HK44: implications for PAH biodegradation in the rhizosphere; Kamath R et al.; The bioluminescent reporter strain Pseudomonas fluorescens HK44 with a nah-lux fusion, was used to investigate the effect of root material (from hybrid poplars, willow, kou, milo, Osage orange, mulberry, and switch grass) and potential root-derived substrates (e.g., sugars, carboxylic acids, amino acids, and phenolics) on the expression of nahG, one of the genes responsible for naphthalene dioxygenase transcription . Whereas nahG was induced by some phenolic substrates that could be released by plants (i.e., salicylate, methyl salicylate, and acetyl salicylate), no induction by root extracts was observed . Rather, increasing root extract concentrations (50 to 275 mg L(-1) as total organic carbon) inhibited nahG expression in assays with cells concurrently exposed to naphthalene . Root extracts also decreased nahG expression at the individual cell level during naphthalene degradation assays . However, treatments with root extracts exhibited significantly higher microbial growth and overall bioluminescence, indicating a higher level of nahG expression by the resulting larger microbial population . This generally resulted in faster naphthalene degradation rates, suggesting that plant-promoted proliferation of competent genotypes could compensate for the interference that labile substrates exert on the expression of genes that code for the degradation of polynuclear aromatic hydrocarbons (PAHs) . This could explain the faster PAH degradation commonly reported in planted than in unplanted soils.

Appl Environ Microbiol, 2004 Apr, 70(4), 1990 - 8
Cross talk between 2,4-diacetylphloroglucinol-producing biocontrol pseudomonads on wheat roots; Maurhofer M et al.; The performance of Pseudomonas biocontrol agents may be improved by applying mixtures of strains which are complementary in their capacity to suppress plant diseases . Here, we have chosen the combination of Pseudomonas fluorescens CHA0 with another well-characterized biocontrol agent, P . fluorescens Q2-87, as a model to study how these strains affect each other's expression of a biocontrol trait . In both strains, production of the antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a crucial factor contributing to the suppression of root diseases . DAPG acts as a signaling compound inducing the expression of its own biosynthetic genes . Experimental setups were developed to investigate whether, when combining strains CHA0 and Q2-87, DAPG excreted by one strain may influence expression of DAPG-biosynthetic genes in the other strain in vitro and on the roots of wheat . DAPG production was monitored by observing the expression of lacZ fused to the biosynthetic gene phlA of the respective strain . Dual-culture assays in which the two strains were grown in liquid medium physically separated by a membrane revealed that Q2-87 but not its DAPG-negative mutant Q2-87::Tn5-1 strongly induced phlA expression in a DeltaphlA mutant of strain CHA0 . In the same way, phlA expression in a Q2-87 background was induced by DAPG produced by CHA0 . When coinoculated onto the roots of wheat seedlings grown under gnotobiotic conditions, strains Q2-87 and CHA0, but not their respective DAPG-negative mutants, were able to enhance phlA expression in each other . In summary, we have established that two nonrelated pseudomonads may stimulate each other in the expression of an antimicrobial compound important for biocontrol . This interpopulation communication occurs in the rhizosphere, i.e., at the site of pathogen inhibition, and is mediated by the antimicrobial compound itself acting as a signal exchanged between the two pseudomonads.

Mol Microbiol, 2004 Apr, 52(2), 371 - 84
The Pseudomonas siderophore quinolobactin is synthesized from xanthurenic acid, an intermediate of the kynurenine pathway; Matthijs S et al.; To cope with iron deficiency fluorescent pseudomonads produce pyoverdines which are complex peptidic siderophores that very efficiently scavenge iron . In addition to pyoverdine some species also produce other siderophores . Recently, it was shown that Pseudomonas fluorescens ATCC 17400 produces the siderophore quinolobactin, an 8-hydroxy-4-methoxy-2-quinoline carboxylic acid (Mossialos, D., Meyer, J.M., Budzikiewicz, H., Wolff, U., Koedam, N., Baysse, C., Anjaiah, V., and Cornelis, P . (2000) Appl Environ Microbiol 66: 487-492) . The entire quinolobactin biosynthetic, transport and uptake gene cluster, consisting out of two operons comprising 12 open reading frames, was cloned and sequenced . Based on the genes present and physiological complementation assays a biosynthetic pathway for quinolobactin is proposed . Surprisingly, this pathway turned out to combine genes derived from the eukaryotic tryptophan-xanthurenic acid branch of the kynurenine pathway and from the pathway for the biosynthesis of pyridine-2,6-bis(thiocarboxylic acid) from P . stutzeri, PDTC . These results clearly show the involvement of the tryptophan-kynurenine-xanthurenic acid pathway in the synthesis of an authentic quinoline siderophore.

Microb Ecol, 2004 Apr, 47(3), 218 - 23 Epub 2004 Apr 02.
Distribution of Nevskia ramosa and other rosette-forming neustonic bacteria; Pladdies T et al.; Samples from 27 natural and artificial aquatic environments were analyzed for the presence of rosette-forming bacteria by a combined cultivation and molecular biological approach . Rosette-forming bacteria developed in 20 enrichment cultures with ammonia-free medium under air . Three morphotypes could be distinguished . The most abundant type I resembled Nevskia ramosa and formed hydrophobic, flat, and dichotomously branching rosettes . Type II rosettes were three-dimensional and were observed in 10 enrichments, often together with those of type I . These rosettes were hydrophilic indicating life in the hyponeuston underneath the air-water interface . Rosettes of a third type consisted of hydrophilic slime stalks that were excreted at the cell poles and were observed in only one sample . Using fluorescence in situ hybridization (FISH) with the Nevskia-specific probes NEV177 and NEV656, the presence of Nevskia ramosa was demonstrated in exactly those samples that showed type I rosettes . In a series of most-probable-number experiments, during a calm and sunny weather period 430,000 Nevskia-like bacteria per mL were found in surface samples, while during rainy weather and within the water body the numbers were lower by several orders of magnitude . Five pure cultures isolated from various enrichments were characterized in detail . The two isolates forming type I rosettes were identified as Nevskia ramosa by 16S rDNA analysis . However, comparison by genomic fingerprinting (ERIC-PCR) revealed differences between the two isolates and previously characterized strains . The 16S rDNA of two isolates forming type II rosettes showed 97.6% similarity to that of Pseudomonas fluorescens . The closest relative of the isolate forming type III rosettes was Sphingomonas parapaucimobilis (96.4% sequence similarity of the 16S rRNA sequence) . All isolates grew homogeneously submersed if ammonia was added to the medium . Our results indicate that Nevskia ramosa is a widely distributed epineustonic bacterium, which can specifically be deleted by its flat and hydrophobic rosettes on ammonia-free media.

Syst Appl Microbiol, 2004 Feb, 27(1), 93 - 108
Phylogenetic relationships between environmental and clinical isolates of Pseudomonas fluorescens and related species deduced from 16S rRNA gene and OprF protein sequences; Bodilis J et al.; The major surface protein of the genus Pseudomonas, OprF, is a non-specific porin that plays an important role in maintenance of cell shape, in growth in a low osmolarity environment, and in adhesion to various supports . The objectives of our study were (i) to carry out a comparative analysis of phylogenies obtained from the OprF protein and from the 16S rRNA gene in 41 isolates from various sources (water, soil, milk and the hospital) and (ii) to investigate the physiological characteristics correlated with the phylogeny of OprF . We report here an important incongruence between the phylogenies of the 16S rRNA gene and the OprF protein . Phylogenetic analysis of 16S rRNA genes grouped Pseudomonas fluorescens isolates into one cluster (termed fluorescens r-cluster) whilst the phylogeny of the OprF protein divided Pseudomonas fluorescens isolates into two quite distinct clusters (termed fluorescens 1 o-cluster and fluorescens 2 o-cluster) that may be related to the original habitat of the strain . The fluorescens 1 o-cluster contained the majority of non-rhizospheric soil isolates, while the fluorescens 2 o-cluster contained all our clinical isolates and most of the rhizospheric isolates (which are fixed to the roots) . In order to check this correlation, we studied two physiological characteristics: the range of growth temperature and the capacity for non-specific adhesion to polystyrene . The temperature range study for strains did not explain the existence of the two o-clusters but it did confirm the capacity of certain P . fluorescens strains to grow at 37 degrees C . The adhesion capacities of the isolates in the two o-clusters seems to be correlated with ecological niche.

Mol Cell Probes, 2004 Feb, 18(1), 67 - 73
Real-time PCR assays for genus-specific detection and quantification of culturable and non-culturable mycobacteria and pseudomonads in metalworking fluids; Khan IU et al.; Genus-specific real-time PCR assays were developed and optimized for the direct culture-independent detection and quantification of Mycobacteria and Pseudomonads in contaminated metalworking fluids (MWF) and the results were compared with conventional culturing using selective media . It included optimization of the direct DNA isolation from the fluid matrix and the amplification conditions using genus-specific primers . Mycobacterium-specific primers based on 65-kDa heat shock protein (hsp) gene, and Pseudomonas-specific primers based on 16S rRNA gene were used . A standard curve was developed each for the two model bacterial species Mycobacterium immunogenum and Pseudomonas fluorescens, representing two important genera frequently isolated from MWF . A minimum quantification limit of 10 cells/ml was achieved although as low as 1 cell/ml yielded a detectable amplicon signal . Of the twenty MWF field samples contaminated with mixed microflora, only two samples yielded putative colonies of Mycobacteria and Pseudomonads by culturing method, while seven samples responded to the genus-specific real-time PCR detection and quantification for each genus . In contrast to the low culturable counts, the real-time PCR based cell counts ranged from 1.3 x 10(2) to 5.5 x 10(5)cells/ml and 5.2 x 10(2) to 7.0 x 10(5)cells/ml for Mycobacteria and Pseudomonads, respectively, indicating a significant non-culturable fraction in the fluids, for the two genera . This is the first application of real-time PCR protocol to MWF samples for detection and quantification of total (culturable and non-culturable) Mycobacteria and Pseudomonads without culturing.

J Biol Chem, 2004 Jun 11, 279(24), 25066 - 74 Epub 2004 Mar 18.
A repeated GGA motif is critical for the activity and stability of the riboregulator RsmY of Pseudomonas fluorescens; Valverde C et al.; The riboregulator RsmY of Pseudomonas fluorescens strain CHA0 is an example of small regulatory RNAs belonging to the global Rsm/Csr regulatory systems controlling diverse cellular processes such as glycogen accumulation, motility, or formation of extracellular products in various bacteria . By binding multiple molecules of the small regulatory protein RsmA, RsmY relieves the negative effect of RsmA on the translation of several target genes involved in the biocontrol properties of strain CHA0 . RsmY and functionally related riboregulators have repeated GGA motifs predicted to be exposed in single-stranded regions, notably in the loops of hairpins . The secondary structure of RsmY was corroborated by in vivo cleavage with lead acetate . RsmY mutants lacking three or five (out of six) of the GGA motifs showed reduced ability to derepress the expression of target genes in vivo and failed to bind the RsmA protein efficiently in vitro . The absence of GGA motifs in RsmY mutants resulted in reduced abundance of these transcripts and in a shorter half-life (< or = 6 min as compared with 27 min for wild type RsmY) . These results suggest that both the interaction of RsmY with RsmA and the stability of RsmY strongly depend on the GGA repeats and that the ability of RsmY to interact with small regulatory proteins such as RsmA may protect this RNA from degradation.

Biochemistry, 2004 Mar 23, 43(11), 3230 - 7
Reaction of Pseudomonas fluorescens kynureninase with beta-benzoyl-L-alanine: detection of a new reaction intermediate and a change in rate-determining step; Gawandi VB et al.; Beta-benzoyl-DL-alanine was synthesized from alpha-bromoacetophenone and diethyl acetamidomalonate . The racemic amino acid was resolved by carboxypeptidase A-catalyzed hydrolysis of the N-trifluoroacetyl derivative . Beta-benzoyl-L-alanine is a good substrate of kynureninase from Pseudomonas fluorescens, with k(cat) and k(cat)/K(m) values of 0.7 s(-1) and 8.0 x 10(4) M(-1) s(-1), respectively, compared to k(cat) = 16.0 s(-1) and k(cat)/K(m) = 6.0 x 10(5) M(-1) s(-1) for L-kynurenine . In contrast to the reaction of L-kynurenine, beta-benzoyl-L-alanine does not exhibit a significant solvent isotope effect on k(cat) ((H)k/(D)k = 0.96 +/- 0.06) . The pre-steady-state kinetics of the reaction of beta-benzoyl-L-alanine were investigated by rapid scanning stopped-flow spectrophotometry . The spectra show the formation of a quinonoid intermediate, with lambda(max) = 490 nm, in the dead time of the instrument, which then decays, with k = 210 s(-1), to form a transient intermediate with lambda(max) at 348 nm . In the presence of benzaldehyde, the 348 nm intermediate decays, with k = 0.7 s(-1), to form a quasistable quinonoid species with lambda(max) = 492 nm . Previous studies demonstrated that benzaldehyde can trap an enamine intermediate formed after the C(beta)-C(gamma) bond cleavage {Phillips, R . S., Sundararaju, B., and Koushik, S . V . (1998) Biochemistry 37, 8783-8789} . Thus, the 348 nm intermediate is kinetically competent . The position of the absorption maximum and shape of the band is consistent with a PMP-ketimine intermediate . The results from chemical quenching analysis do not show a burst of benzoate and, thus, also support the formation of benzoate as the rate-determining step . These data suggest that, in contrast to L-kynurenine, for which the rate-determining step was shown to be deprotonation of the pyruvate-ketimine intermediate {Koushik, S . V., Moore, J . A., III, Sundararaju, B., and Phillips, R . S . (1998) Biochemistry 37, 1376-1382}, the rate-determining step in the reaction of beta-benzoyl-L-alanine with kynureninase is C(beta)-C(gamma) bond cleavage.

Appl Environ Microbiol, 2004 Mar, 70(3), 1836 - 42
Potential role of pathogen signaling in multitrophic plant-microbe interactions involved in disease protection; Duffy B et al.; Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease . Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions . Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions . We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f . sp . radicis-lycopersici on the tomato as determined with mutational analysis . In contrast, DAPG was not important for the less-disease-suppressive strain CHA0 . This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87 . In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.

Appl Environ Microbiol, 2004 Mar, 70(3), 1758 - 66
Positive autoregulation and signaling properties of pyoluteorin, an antibiotic produced by the biological control organism Pseudomonas fluorescens Pf-5; Brodhagen M et al.; Pseudomonas fluorescens Pf-5, a rhizosphere bacterium, produces a suite of secondary metabolites that are toxic to seed- and root-rotting plant pathogens . Among these are the polyketide compounds pyoluteorin and 2,4-diacetylphloroglucinol . We provide evidence that pyoluteorin production is influenced by positive autoregulation . Addition of pyoluteorin to liquid cultures of Pf-5 enhanced pyoluteorin production . In addition, pyoluteorin and 2,4-diacetylphloroglucinol mutually inhibit one another's production in Pf-5 . For pyoluteorin, both positive autoregulation and negative influences on production by 2,4-diacetylphloroglucinol were demonstrated at the transcriptional level by measuring activity from transcriptional fusions of an ice nucleation reporter gene (inaZ) to three separate pyoluteorin biosynthetic genes . The occurrence of pyoluteorin autoregulation in the rhizosphere was assessed on cucumber seedlings in pasteurized soil with cross-feeding experiments . In the rhizosphere, expression of a pyoluteorin biosynthesis gene by a pyoluteorin-deficient mutant of Pf-5 was enhanced by pyoluteorin produced by coinoculated cells of Pf-5 . These data establish that the polyketide pyoluteorin is an autoregulatory compound and functions as a signal molecule influencing the spectrum of secondary metabolites produced by the bacterial cell.

Proc R Soc Lond B Biol Sci, 2004 Jan 7, 271(1534), 107 - 11
The effect of spatial heterogeneity and parasites on the evolution of host diversity; Brockhurst MA et al.; Both spatial heterogeneity and exploiters (parasites and predators) have been implicated as key ecological factors driving population diversification . However, it is unclear how these factors interact . We addressed this question using the common plant-colonizing bacterium Pseudomonas fluorescens, which has been shown to diversify rapidly into spatial niche-specialist genotypes when propagated in laboratory microcosms . Replicate populations were evolved in spatially homogeneous and heterogeneous environments (shaken and static microcosms, respectively) with and without viral parasites (bacteriophage) for approximately 60 bacterial generations . Consistent with previous findings, exploiters reduced diversity in heterogeneous environments by relaxing the intensity of resource competition . By contrast, exploiters increased diversity in homogeneous environments where there was little diversification through resource competition . Competition experiments revealed this increase in diversity to be the result of fitness trade-offs between exploiter resistance and competitive ability . In both environments, exploiters increased allopatric diversity, presumably as a result of divergent selection for resistance between populations . Phage increased total diversity in homogeneous environments, but had no net effect in heterogeneous environments . Such interactions between key ecological variables need to be considered when addressing diversification and coexistence in future studies.

Microbiology, 2004 Mar, 150(Pt 3), 657 - 64
Smc01944, a secreted peroxidase induced by oxidative stresses in Sinorhizobium meliloti 1021; Barloy-Hubler F et al.; Sequencing of the Sinorhizobium meliloti strain 1021 genome led to the detection of 6204 open reading frames, 41 % of which have no hypothetical function . To help annotate this genome, a transcriptome analysis was carried out with a dedicated microarray consisting of 146 genes belonging to three different classes: (i) no hypothetical function; (ii) potentially involved in oxidative stress responses; (iii) known to participate in oxidative stress responses (e.g . catalase and superoxide dismutase genes) . This transcriptome analysis, together with biological experiments and in silico investigations, identified new genes induced by exogenous H(2)O(2) . The smc01944 gene was the most strongly induced: quantitative PCR showed that the amount of smc01944 mRNA increased 50-fold following the addition of 10 mM H(2)O(2), whereas the amount of katA mRNA (encoding a catalase) only increased 10-fold . Smc01944 is a non-haem chloroperoxidase (Cpo) . The only member of this family to have been so far characterized is encoded by prxC of Pseudomonas fluorescens . Unexpectedly, the NH(2)-terminus of Smc01944 includes a signal peptide and Smc01944 is secreted into the supernatant . Interestingly, smc01944 is preceded by smc01945, encoding an OhrR-like regulator (MarR family) . Thus, Smc01944 is the first exported Cpo encoded by a gene possibly regulated by an OhrR regulator . It was also shown that smc01944 is induced by t-butyl and cumene hydroperoxides but only slightly by menadione . The study of Smc01944 described in this work showed that the oxidative stress response of S . meliloti seems to differ from that of other bacteria characterized to date.

Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 3269 - 74 Epub 2004 Feb 23.
Pseudomonas syringae pv . tomato cells encounter inhibitory levels of water stress during the hypersensitive response of Arabidopsis thaliana; Wright CA et al.; During plant defense against bacterial pathogens, the hypersensitive response (HR) functions to restrict pathogen growth and spread . The mechanisms driving this growth restriction are poorly understood . We used a water stress-responsive transcriptional fusion to quantify the water potential sensed by individual Pseudomonas syringae pv . tomato DC3000 cells during infection of Arabidopsis thaliana leaves . A nonpathogenic DC3000 hrcC mutant defective in type III secretion, as well as the saprophyte Pseudomonas fluorescens A506, sensed water potentials of -0.3 to -0.4 MPa at 48 h postinfiltration (hpi) . During pathogenesis, DC3000 sensed lower water potentials (-0.4 to -0.9 MPa), demonstrating that it can modify the intercellular environment, and these water potentials were associated with optimal DC3000 growth in culture . During the HR, DC3000 cells sensed water potentials (-1.6 to -2.2 MPa) that were low enough to prevent cell division in the majority of cells in culture . This water potential decrease occurred within only 4 hpi and was influenced by avirulence gene expression, with avrRpm1 expression associated with lower water potentials than avrRpt2 or avrB expression at 48 hpi . The population sizes of the DC3000 variants tested were significantly correlated with the apoplastic water potential at 48 hpi, with a decrease of -0.9 MPa associated with a 10-fold decrease in cells per gram of leaf . These results suggest that the apoplastic water potential is a determinant of endophytic bacterial population size, and water stress, resulting from high osmolarity or tissue desiccation, is at least one factor restricting bacterial growth during the HR.

J Appl Microbiol, 2004, 96(3), 437 - 46
The use of a marked strain of Pseudomonas fluorescens to model the spread of brain tissue to the musculature of cattle after shooting with a captive bolt gun; Prendergast DM et al.; AIMS: The aim of this study was to use a marked strain of Pseudomonas fluorescens to model the spread of central nervous system (CNS) tissue in cattle following captive bolt stunning . METHODS AND RESULTS: The marked organism was introduced by injection through the captive bolt aperture immediately after stunning and was subsequently detected in a wide range of derived tissues, including blood, organs, and the musculature of the entire forequarters of test animals . This was dependent on the use of high concentrations of the organism that were recovered sufficiently and rapidly to minimize the bactericidal properties of the circulatory system . These results suggest that a marked organism could potentially be used to model the effects of captive bolt stunning on the dissemination of CNS tissue from the brain . CONCLUSIONS: These results indicate that current commercial methods of captive bolt stunning may induce widespread and significant mobilization of CNS tissue within beef carcasses . This may lead to the widespread dissemination of such materials within meat destined for human consumption . SIGNIFICANCE AND IMPACT OF THE STUDY: In the absence of rapid, simple and sufficiently sensitive methods for the direct detection of prion in commercially slaughtered animals, marked organisms can provide useful models in studies of the dissemination kinetics of prion disease in captive bolt stunned animals.

Mar Biotechnol (NY), 2002 Jun, 4(3), 310 - 22
Production of transgenic medaka with increased resistance to bacterial pathogens; Sarmasik A et al.; Cecropins, first identified in silk moth (Hyalophora cecropia), are a group of antimicrobial peptides with bactericidal activity against a broad spectrum of bacteria . In this study we investigated whether (1) this group of antimicrobial peptides could exhibit bactericidal activity toward known fish bacterial pathogens and (2) expression of cecropin transgenes in transgenic medaka (Oryzias latipas) could result in increasing resistance of the transgenic fish to infection by fish bacterial pathogens . Cecropin gene construct containing silk moth preprocecropin B, procecropin B and cecropin B, and porcine cecropin P1 driven by a cytomegalovirus (CMV) promoter were transfected into chinook salmon embryonic cells (CHSE-214) by lipofection, and the resulting permanent transformants were collected . In an "inhibition zone" assay medium isolated from each transformant exhibited strong bactericidal activity toward known fish bacterial pathogens such as Pseudomonas fluorescens, Aeromonas hydrophila, and Vibrio anguillarum . The same cecropin transgene constructs were introduced into newly fertilized medaka eggs by electroporation to produce transgenic fish . About 40% to 60% of the embryos survived from electroporation, and about 5% to 11% of the surviving fish were shown to contain cecropin transgenes by polymerase chain reaction analysis of genomic DNA samples isolated from presumptive transgenic fish . These P1 transgenic fish were used as founder stocks, and following generations of successive breeding, a total of 20 F2 families of transgenic fish were established . Expression of cecropin transgenes was detected in the F2 transgenics by reverse transcriptase polymerase chain reaction analysis . Southern blot analysis of genomic DNA isolated from different F2 fish showed that cecropin transgenes were integrated into the genomes of F2 transgenic fish . To determine whether transgenic fish carrying cecropin transgenes could exhibit resistance to infection by known fish bacterial pathogens, F2 transgenic fish from different families and control fish were challenged with P . fluorescens and V . anguillarum at a 60% lethal dose . Challenge studies showed that while about 40% of the control fish were killed by both pathogens, only up to 10% of the F2 transgenic fish were killed by P . fluorescens and about 10% to 30% by V . anguillarum . These results clearly showed that the transgenic medaka carrying cecropin transgenes had acquired elevated resistance to bacterial infection.

Appl Environ Microbiol, 2004 Feb, 70(2), 850 - 4
Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria; Haddix PL et al.; The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water . We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria . Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample . Pseudomonas fluorescens P-17 and Spirillum sp . strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium . Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity . All mutants tested were able to grow in tap water under AOC assay conditions . Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test . Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring . Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay . Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability . This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.

Poult Sci, 2004 Jan, 83(1), 95 - 100
Microbial contamination in inoculated shell eggs: II . Effects of layer strain and egg storage; Jones DR et al.; Three Ottawa control strains and a current commercial laying stock were reared and housed in the same environment . Eggs were collected at 5 different hen ages throughout the 2 production cycles of the flock . The eggs were inoculated with Salmonella Enteritidis (SE), Pseudomonas fluorescens (PF), a combination of the 2, or sterile buffered peptone water and stored up to 5 wk . After storage at room temperature, contamination levels were determined for the exterior surface, air cell, egg contents, and within the shell . Interior, egg contents, and shell contamination levels of SE and PF increased with storage time . There were no apparent increases in the infectivity of SE or PF in the presence of the other organism . PF was a poor survivor on the shell surface under these storage conditions . Throughout the 5-wk storage, eggs from control strain 10 maintained their microbial integrity more effectively . Eggs from control strain 5 and the current commercial stock were more easily contaminated than the other strains . These data suggest that genetic selection has altered microbiological defenses of the eggs produced.

Biochemistry, 2004 Feb 10, 43(5), 1193 - 203
Three-dimensional structure of kynureninase from Pseudomonas fluorescens; Momany C et al.; Kynureninase {E.C . 3.7.1.3} is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the hydrolytic cleavage of l-kynurenine to anthranilic acid and l-alanine . Sequence alignment with other PLP-dependent enzymes indicated that kynureninase is in subgroup IVa of the aminotransferases, along with nifS, CsdB, and serine-pyruvate aminotransferase, which suggests that kynureninase has an aminotransferase fold . Crystals of Pseudomonas fluorescens kynureninase were obtained, and the structure was solved by molecular replacement using the CsdB coordinates combined with multiple isomorphous heavy atom replacement . The coordinates were deposited in the PDB (ID code 1QZ9) . The structure, refined to an R factor of 15.5% to 1.85 A resolution, is dimeric and has the aminotransferase fold . The structure also confirms the prediction from sequence alignment that Lys-227 is the PLP-binding residue in P . fluorescens kynureninase . The conserved Asp-201, expected for an aminotransferase fold, is located near the PLP nitrogen, but Asp-132 is also strictly conserved and at a similar distance from the pyridinium nitrogen . Mutagenesis of both conserved aspartic acids shows that both contribute equally to PLP binding, but Asp-201 has a greater role in catalysis . The structure shows that Tyr-226 donates a hydrogen bond to the phosphate of PLP . Unusual among PLP-dependent enzymes, Trp-256, which is also strictly conserved in kynureninases from bacteria to humans, donates a hydrogen bond to the phosphate through the indole N1-hydrogen.

Curr Microbiol, 2003 Dec, 47(6), 521 - 7
Adaptation of Pseudomonas fluorescens to Al-citrate: involvement of tricarboxylic acid and glyoxylate cycle enzymes and the influence of phosphate; Appanna VD et al.; The degradation of Aluminum-citrate by Pseudomonas fluorescens necessitated a major restructuring of the various enzymatic activities involved in the TCA and glyoxylate cycles . While a six-fold increase in fumarase (FUM EC 4.2.1.2) activity was observed in cells subjected to Al-citrate compared to control cells, citrate synthase (CS EC 4.1.3.7) activity experienced a two-fold increase . On the other hand, in the Al-stressed cells malate synthase (MS EC 4.1.3.2) activity underwent a five-fold decrease in activity . This modulation of enzymatic activities appeared to be evoked by Al stress, as the incubation of Al-stressed cells in control media led to the complete reversal of these enzymatic profiles . These observations were further confirmed by 1H NMR and 13C NMR spectroscopy . No significant variations were observed in the activities of other glyoxylate and TCA cycle enzymes, like isocitrate lyase (ICL EC 4.1.3.1), malate dehydrogenase (MDH EC 1.1.1.37), and succinate dehydrogenase (SDH EC 1.3.99.1) . This reconfiguration of the metabolic pathway appears to favour the production of a citrate-rich aluminophore that is involved in the sequestration of Al.

Res Microbiol, 2004 Jan-Feb, 155(1), 39 - 46
Regulation of the cytotoxic effects of Pseudomonas fluorescens by growth temperature; Picot L et al.; We had previously shown that the psychrotrophic bacterium Pseudomonas fluorescens can act as a pathogen, inducing apoptosis and necrosis in neurons and glial cells . In the present study, we investigated the influence of the growth temperature of P . fluorescens on its infectious potential . Adherence of P . fluorescens to glial cells was found to be maximal with bacteria grown at a low temperature (8 degrees C) . At that temperature the swimming behaviour was markedly reduced . An increase in the growth temperature to 19, 28 or 32 degrees C strongly diminished the binding of bacteria to host cells . Thus, the adhesion phenotype of P . fluorescens appears to be independent of the motility of the bacteria . The apoptotic effect of P . fluorescens, determined by morphological (nuclear condensation) and biochemical (induction of nitric oxide synthase activity) indicators, correlated well with its binding activity on glial cells . In contrast, there was a clear dissociation between maximum binding and maximal necrotic action (measured by the release of lactate dehydrogenase) observed with bacteria grown at 19 degrees C . As suggested by capillary electrophoresis analysis, the differences in apoptotic effects may be related to variations in the molecular structure of LPS originating from bacteria grown at low and high temperatures, whereas the necrotic effect, which was maximal at the optimum temperature for the secretion of exoenzymes, could reflect variations in the metabolic activity of bacteria.

Lett Appl Microbiol, 2004, 38(2), 169 - 75
Trichoderma harzianum enhances the production of nematicidal compounds in vitro and improves biocontrol of Meloidogyne javanica by Pseudomonas fluorescens in tomato; Siddiqui IA et al.; AIMS: To determine the influence of soil-borne fungus Trichoderma harzianum on the biocontrol performance of Pseudomonas fluorescens strain CHA0 and its 2,4-diacetylphloroglucinol (DAPG) overproducing derivative CHA0/pME3424 against Meloidogyne javanica . METHODS AND RESULTS: Amendment of the culture filtrate (CF) or methanol extract of the CF of a T . harzianum strain Th6 to P . fluorescens growth medium enhanced the production of nematicidal compound(s) by bacterial inoculants in vitro . In addition, bacteria overwhelmingly expressed phl'-'lacZ reporter gene when the medium was amended with CF of T . harzianum . Pseudomonas fluorescens and T . harzianum applied together in unsterilized sandy loam soil caused greater reduction in nematode population densities in tomato roots . CONCLUSIONS: Trichoderma harzianum improves root-knot nematode biocontrol by the antagonistic rhizobacterium P . fluorescens both in vitro and under glasshouse conditions . SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic effect of T . harzianum on the production of nematicidal compound(s) critical in biocontrol may improve the efficacy of biocontrol bacteria against plant-parasitic nematodes . Considering the inconsistent performance of the biocontrol agents under field conditions, application of a mixture of compatible T . harzianum and P . fluorescens would more closely mimic the natural situation and might broaden the spectrum of biocontrol activity with enhanced efficacy and reliability of control.

Biochem Biophys Res Commun, 2004 Feb 13, 314(3), 897 - 901
Bacteriocin of Enterococcus from lactoserum able to cause oxidative stress in Staphylococcus aureus; Eraso AJ et al.; The effect of a bacteriocin of Enterococcus on the oxidative metabolism of sensitive bacteria was investigated through the detection of oxidative stress by chemiluminescence (CL) . The bacteriocin named EntB was purified to study the action on Staphylococcus aureus isolated from cosmetic . Chromatographic separation of EntB indicated different states of oligomerization with molecular weights multiple of 12,000Da monomeric form . The monomer purified by ion exchange was studied in its capacity to affect the oxidative metabolism of S . aureus, which showed increase of anion superoxide (O(2)(-)) when incubated with EntB . This effect was compared to the action of EntB on leukocytes as an assay of toxicity . EntB did not generate significant oxidative stress in leukocytes . Pyoverdin, a leukotoxic pigment of Pseudomonas fluorescens, was taken as reference, and it was found that this pigment caused similar oxidative stress to EntB in S . aureus; however, pyoverdin generated high production of anion superoxide (O(2)(-)) in leukocytes, while EntB did not increase the level of O(2)(-).

Bioorg Med Chem Lett, 2004 Feb 9, 14(3), 585 - 9
Chemoenzymatic synthesis and binding affinity of novel (R)- and (S)-3-aminomethyl-1-tetralones, potential atypical antipsychotics; Caro Y et al.; A series of (R)- and (S)-3-aminomethyl-1-tetralones, conformationally constrained analogues of haloperidol, have been obtained by enzymatic resolution of the corresponding racemic 3-hydroxymethyl-1-tetralones using Pseudomonas fluorescens lipase . Their binding affinities at dopamine D(2) and serotonin 5-HT(2A) and 5-HT(2C) receptors were determined showing in some cases an atypical antipsychotic profile with Meltzer's ratio higher than 1.30.

J Biotechnol, 2004 Feb 5, 107(3), 255 - 63
Degradation of alpha-pinene oxide and {2H7}-2,5,6-trimethyl-hept-(2E)-enoic acid by Pseudomonas fluorescens NCIMB 11761; Zorn H et al.; When submerged cultured Pseudomonas fluorescens NCIMB 11761 was fed-batch supplemented with alpha-pinene oxide, a rapid formation of 2,6-dimethyl-5-methylene-hept-(2Z)-enal (I) (isonovalal) was observed . Biotransformation and isomerisation of (I) to the (2E)-isomer (II) (novalal) were enhanced by Lewatit OC 1064, a macroporous polystyrene adsorbent . Accelerated isomerisation in the presence of an amino donor (glycine) at pH 7.3 pointed to a merely chemical mechanism . A maximum yield of 48 g of aldehydesl(-1) was achieved, but quantitative analysis of the volatile fraction showed that the molar conversion of the pinene oxide substrate reached no more than 67% . To fill this gap of the mass balance, the acidic fraction was isolated . It contained several compounds which suggested a beta-oxidation-like catabolism starting from 2,6-dimethyl-5-methylene-hept-(2E)-enoic acid (III) (novalic acid) . Using {2H7}-2,5,6-dimethyl-hept-(2E)-enoic acid as a conversion substrate and gas chromatography coupled to atomic emission detection and mass spectrometry a degradation pathway via labelled 3,4-dimethylpentenoic and methylpropanoic acids was evidenced . This pathway may play a predominant role in isoprenoid degradation by soil bacteria.

Ying Yong Sheng Tai Xue Bao, 2003 Sep, 14(9), 1585 - 7
{Effect of bacterivorous nematodes on bacteria population under gnotobiotic culture}; Chen X et al.; A gnotobiotic microcosm experiment was conducted to study the influence of bacterivorous nematodes (Protorhabditis sp.) on bacteria (Pseudomonas fluorescens) population under different conditions including substrate concentration, oscillation pattern, and numbers of nematodes inoculated . When the bacteria were incubated under intermittent oscillation by hand (6 times at 0.5 h intervals, 22 degrees C), their growth was stimulated in the presence of nematoedes, and the bacteria grew faster with the increase of nematode numbers and substrate (liquid potato-sucrose medium) concentration . However, when the incubation was under continuous oscillation (100 rpm, 22 degrees C), bacteria population was deflated with the addition of nematodes, and the inhibition was greater when a higher concentration of substrate was used . It was found that the stimulation or inhibition of bacteria population by nematodes occurred in the logarithmic stage of bacteria growth . The optimal and over-grazing of nematodes on regulating bacteria population was discussed.

Biochemistry, 2004 Jan 27, 43(3), 663 - 74
Engineering p-hydroxyphenylpyruvate dioxygenase to a p-hydroxymandelate synthase and evidence for the proposed benzene oxide intermediate in homogentisate formation; Gunsior M et al.; p-Hydroxyphenylpyruvate dioxygenase (HPD) plays a key role in the normal catabolism of tyrosine . An Fe2+/oxygen-dependent enzyme, it converts p-hydroxyphenylpyruvate into homogentisate and is part of the superfamily of alpha-ketoglutarate-dependent enzymes that couples oxidative decarboxylation of an alpha-ketoacid cofactor to oxidative modification of its substrate . In this case, the alpha-ketoacid is part of the substrate side chain . HPD shows strong homology to p-hydroxymandelate synthase (HMS), an enzyme that catalyzes the formation of p-hydroxymandelate from p-hydroxyphenylpyruvate, an early step in the biosynthesis of p-hydroxyphenylglycine, which is a nonproteinogenic amino acid incorporated into several biologically active secondary metabolites . Sequence alignment between the HPD and the HMS enzyme families and analysis of the Pseudomonas fluorescens HPD crystal structure highlighted four residues within each active site that may play roles in catalytic differentiation between the two products . We attempted to convert Streptomyces avermitilis HPD into an engineered S . avermitilis HMS by site-directed mutagenesis of these four residues individually and in combination . HPLC assay analysis of each His6-tagged mutant indicated that F337I successfully produced p-hydroxymandelate, along with homogentisate and an unknown compound . The structure of the latter was determined to be an oxepinone derived from the benzene-oxide intermediate long hypothesized in HPD catalysis.

Microbiol Res, 2003, 158(4), 359 - 62
'P' solubilization potential of plant growth promoting Pseudomonas mutants at low temperature; Das K et al.; Pseudomonas fluorescens strain GRS1, PRS9 and their cold tolerant mutants were examined for their tricalcium phosphate (TCP) solubilizing activity in NBRIP (broth) media at 10 degrees C and 25 degrees C . Invariably, all the cold tolerant mutants of GRS1 and PRS9 were found more efficient than their respective wild type counterparts for 'P' solubilization activity at 10 degrees C as compared to 25 degrees C . 'P' solubilization potential of CRM was found maximum among all the strains followed by CRPF6 and CRPF4 . To the best of out knowledge, this is the first report regarding low temperature 'P' solubilization activity.

Appl Environ Microbiol, 2004 Jan, 70(1), 121 - 8
Enzymatic assimilation of cyanide via pterin-dependent oxygenolytic cleavage to ammonia and formate in Pseudomonas fluorescens NCIMB 11764; Fernandez RF et al.; Utilization of cyanide as a nitrogen source by Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, with the latter compound satisfying the nitrogen requirement . Substrate attack is initiated by cyanide oxygenase (CNO), which has been shown previously to have properties of a pterin-dependent hydroxylase . CNO was purified 71-fold and catalyzed the quantitative conversion of cyanide supplied at micromolar concentrations (10 to 50 micro M) to formate and ammonia . The specific activity of the partially purified enzyme was approximately 500 mU/mg of protein . The pterin requirement for activity could be satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 micro M) . These compounds included, for example, biopterin, monapterin, and neopterin, all of which were also identified in cell extracts . Substrate conversion was accompanied by the consumption of 1 and 2 molar equivalents of molecular oxygen and NADH, respectively . When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted and displayed an overall reaction stoichiometry of 1:1:1 for cyanide, O(2), and NADH consumed . Cyanide was also attacked by CNO at a higher concentration (1 mM), but in this case formamide accumulated as the major reaction product (formamide/formate ratio, 0.6:0.3) and was not further degraded . A complex reaction mechanism involving the production of isocyanate as a potential CNO monooxygenation product is proposed . Subsequent reduction of isocyanate to formamide, whose hydrolysis occurs as a CNO-bound intermediate, is further envisioned . To our knowledge, this is the first report of enzymatic conversion of cyanide to formate and ammonia by a pterin-dependent oxygenative mechanism.

Microbiol Immunol, 2003, 47(12), 895 - 901
Characterization and localization of fluorescent Pseudomonas cold shock protein(s) by monospecific polyclonal antibodies; Khan M et al.; Cold shock protein (CSP) from Pseudomonas fluorescens MTCC 103 and cold resistant protein (CRP) from its mutant CRPF8 of 14 and 35 kd, respectively were purified to homogeneity by HPLC . Polyclonal antibodies were raised against these proteins and the expression level was checked at different temperatures, i.e., 4, 10, 20, 30 and 37 C . Furthermore, morphological changes in P . fluorescens MTCC 103 and its mutant (CRPF8) were analyzed by transmission electron microscopy (TEM) . Localization of CSP and CRP documented with immunoelectron microscopy, using colloidal gold particles conjugated with secondary antibodies being the probe were used . Nevertheless, the results of cytosolic localization of CSP and CRP were evident . Furthermore, the expression of CSP and CRP increased with decrease in temperature and the cell wall thickness of the mutant exhibited 2-fold increase, thus facilitating low temperature survival.

Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 184 - 6 Epub 2003 Dec 18.
The purification, crystallization and preliminary structural characterization of PhzF, a key enzyme in the phenazine-biosynthesis pathway from Pseudomonas fluorescens 2-79; Mavrodi DV et al.; Phenazines produced by members of several bacterial genera are biologically active metabolites that function in microbial competitiveness, the suppression of soil-borne plant diseases and virulence in infectious disease . Despite recent progress towards understanding the biochemistry of phenazine synthesis, the key reactions leading to the formation of the heterocyclic scaffold common to all phenazine compounds remain obscure . Pseudomonas fluorescens 2-79 contains seven phenazine (phz) genes that encode components of the pathway for biosynthesis of phenazine-1-carboxylic acid . A central step in this pathway involves the condensation of two identical precursor molecules derived from chorismic acid and is catalysed by the product of the phzF gene . In this study, recombinant PhzF was purified and crystallized from PEG 4000/ammonium sulfate/sodium citrate pH 5.6 . The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 56.3, c = 156.4 A . They contain one monomer in the asymmetric unit and diffract to better than 1.7 A on synchrotron beamlines . Crystals of seleno-L-methionine-labelled PhzF have been obtained and SAD data are reported.

Science, 2003 Dec 19, 302(5653), 2107 - 9
Adaptation limits diversification of experimental bacterial populations; Buckling A et al.; Adaptation to a specific niche theoretically constrains a population's ability to subsequently diversify into other niches . We tested this theory using the bacterium Pseudomonas fluorescens, which diversifies into niche specialists when propagated in laboratory microcosms . Numerically dominant genotypes were allowed to diversify in isolation . As predicted, populations increased in fitness through time but showed a greatly decreased ability to diversify . Subsequent experiments demonstrated that niche generalists and reductions in intrinsic evolvability were not responsible for our data . These results show that niche specialization may come with a cost of reduced potential to diversify.

FEMS Microbiol Lett, 2003 Dec 12, 229(2), 231 - 6
Branching of o-nitrobenzoate degradation pathway in Arthrobacter protophormiae RKJ100: identification of new intermediates; Pandey G et al.; We have earlier reported a novel reductive pathway for o-nitrobenzoate (ONB) degradation (at 0.5 mM) in Arthrobacter protophormiae RKJ100, which proceeds via the formation of o-hydroxylaminobenzoate (HABA) and anthranilate (AA) . During growth of this organism at 40 times higher concentration (20 mM) of ONB, 3-hydroxyanthranilate (HAA) was identified as an intermediate by thin layer chromatography, gas chromatography and high performance liquid chromatography studies . Crude cell extracts of ONB-grown cells showed HAA 3,4-dioxygenase activity suggesting HAA as a terminal aromatic intermediate of the catabolic energy-yielding pathway as shown before in Pseudomonas fluorescens strain KU-7 . HAA is further cleaved to 2-amino-3-carboxymuconic-6-semialdehyde by the action of HAA 3,4-dioxygenase . In this report we propose that ONB degradation occurs via the formation of HABA and the pathway branches at this point to form the two different aromatic intermediates AA and HAA by the action of a reductase and a mutase, respectively.

Int J Food Microbiol, 2004 Jan 1, 90(1), 63 - 74
Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin; Branen JK et al.; A microtiter plate assay was employed to systematically assess the interaction between ethylenediaminetetraacetic acid (EDTA) or lactoferrin and nisin, lysozyme, or monolaurin against strains of Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, and Pseudomonas fluorescens . Low levels of EDTA acted synergistically with nisin and lysozyme against L . monocytogenes but EDTA and monolaurin interacted additively against this microorganism . EDTA synergistically enhanced the activity of nisin, monolaurin, and lysozyme in tryptic soy broth (TSB) against two enterohemorrhagic E . coli strains . In addition, various combinations of nisin, lysozyme, and monolaurin with EDTA were bactericidal to some gram-negative bacteria whereas none of the antimicrobials alone were bactericidal . Lactoferrin alone (2000 microg ml(-1)) did not inhibit any of the bacterial strains, but did enhance nisin activity against both L . monocytogenes strains . Lactoferrin in combination with monolaurin inhibited growth of E . coli O157:H7 but not E . coli O104:H21 . While lactoferrin combined with nisin or monolaurin did not completely inhibit growth of the gram-negative bacteria, there was some growth inhibition . All combinations of EDTA or lactoferrin with antimicrobials were less effective in 2% fat UHT milk than in TSB . S . enteritidis and P . fluorescens strains were consistently more resistant to antimicrobial combinations . Resistance may be due to differences in the outer membrane and/or LPS structure.

Microbiology, 2003 Dec, 149(Pt 12), 3543 - 52
Co-ordination of iron acquisition, iron porphyrin chelation and iron-protoporphyrin export via the cytochrome c biogenesis protein CcmC in Pseudomonas fluorescens; Baysse C et al.; The cytoplasmic membrane protein CcmC is, together with other Ccm proteins, a component for the maturation of c-type cytochromes in Gram-negative bacteria . A Pseudomonas fluorescens ATCC 17400 ccmC mutant is cytochrome c-deficient and shows considerably reduced production of the two siderophores pyoverdine and quinolobactin, paralleled by a general inability to utilize various iron sources, with the exception of haem . The ccmC mutant accumulates in a 5-aminolevulinic acid-dependent synthesis a reddish, fluorescent pigment identified as protoporphyrin IX . As a consequence a visA phenotype similar to that of a ferrochelatase-deficient hemH mutant characterized by drastically reduced growth upon light exposure was observed for the ccmC mutant . The defect of iron-protoporphyrin formation was further demonstrated by the failure of ccmC cell-free proteinase K-treated extracts to stimulate the growth of a haem auxotrophic hemH indicator strain, compared to similarly prepared wild-type extracts . In addition, the ccmC mutant did not sustain hemH growth in cross-feeding experiments while the wild-type did . Significantly reduced resistance to oxidative stress mediated by haem-containing catalases was observed for the ccmC mutant . A double hemH ccmC mutant could not be obtained in the presence of external haem without the hemH gene in trans, indicating that the combination of the two mutations is lethal . It was concluded that CcmC, apart from its known function in cytochrome c biogenesis, plays a role in haem biosynthesis . A function in the regulatory co-ordination of iron acquisition via siderophores, iron insertion into porphyrin via ferrochelatase and iron-protoporphyrin export for cytochrome c formation is predicted.

Appl Environ Microbiol, 2003 Dec, 69(12), 7161 - 72
Biochemical, genetic, and zoosporicidal properties of cyclic lipopeptide surfactants produced by Pseudomonas fluorescens; De Souza JT et al.; Zoospores play an important role in the infection of plant and animal hosts by oomycetes and other zoosporic fungi . In this study, six fluorescent Pseudomonas isolates with zoosporicidal activities were obtained from the wheat rhizosphere . Zoospores of multiple oomycetes, including Pythium species, Albugo candida, and Phytophthora infestans, were rendered immotile within 30 s of exposure to cell suspensions or cell culture supernatants of the six isolates, and subsequent lysis occurred within 60 s . The representative strain SS101, identified as Pseudomonas fluorescens biovar II, reduced the surface tension of water from 73 to 30 mN m-1 . The application of cell suspensions of strain SS101 to soil or hyacinth bulbs provided significant protection against root rot caused by Pythium intermedium . Five Tn5 mutants of strain SS101lacked the abilities to reduce the surface tension of water and to cause lysis of zoospores . Genetic characterization of two surfactant-deficient mutants showed that the transposons had integrated into condensation domains of peptide synthetases . A partially purified extract from strain SS101 reduced the surface tension of water to 30 mN m-1 and reached the critical micelle concentration at 25 micrograms ml-1 . Reverse-phase high-performance liquid chromatography yielded eight different fractions, five of which had surface activity and caused lysis of zoospores . Mass spectrometry and nuclear magnetic resonance analyses allowed the identification of the main constituent as a cyclic lipopeptide (1,139 Da) containing nine amino acids and a 10-carbon hydroxy fatty acid . The other four zoosporicidal fractions were closely related to the main constituent, with molecular massesranging from 1,111 to 1,169 Da.

J Environ Sci Health B, 2003 Nov, 38(6), 737 - 46
Biodegradation of chlorsulfuron and metsulfuron-methyl by Aspergillus niger in laboratory conditions; Boschin G et al.; Two sulfonylurea herbicides, chlorsulfuron and metsulfuron-methyl, were studied under laboratory conditions, in order to elucidate the biodegradation pathway operated by Aspergillus niger, a common soil fungus, which is often involved in the degradation of xenobiotics . HPLC-UV was used to study the kinetic of degradation, whereas LC-MS was used to identify the metabolites structure . In order to avoid the chemical degradation induced by a decrease in pH, due to the production of citric acid by the fungus, the experiments were performed in a buffered neutral medium . No significant degradation for both compounds was observed in mineral medium with 0.2% sodium acetate . On the contrary, in a rich medium, after 28 days the degradations, chemical degradation excluded, were about 30% for chlorsulfuron and 33% for metsulfuron-methyl . The main microbial metabolites were obtained via cleavage of the sulfonylurea bridge . In addition the fungus seems to be able to hydroxylate the aromatic ring of chlorsulfuron . In the case of metsulfuron-methyl the only detected metabolite was the triazine derivative, while the aromatic portion was completely degraded . Finally, the demethylation of the methoxy group on the triazine ring, previously observed with a Pseudomonas fluorescens strain, was not observed with A . niger.

J Appl Microbiol, 2003, 95(5), 1039 - 48
Impact of biocontrol agents Pseudomonas fluorescens CHA0 and its genetically modified derivatives on the diversity of culturable fungi in the rhizosphere of mungbean; Shaukat SS et al.; AIMS: To assess whether Pseudomonas fluorescens strain CHA0 and its genetically modified derivatives, CHA0/pME3424 (antibiotic over-producer) and CHA89 (antibiotic-deficient) could have an impact on the fungal community structure and composition in the rhizosphere of mungbean . METHODS AND RESULTS: Under glasshouse conditions, mungbean was grown repeatedly in the same soil, which was inoculated with CHA0, CHA0/pME3424, CHA89 or was left untreated . Treatments were applied to soil at the start of each 36-day mungbean growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first, second and third cycles . The effects of CHA0 and CHA0/pME3424 did differ from the controls while CHA89 did not . Whereas all major fungal species were frequently isolated from both bacterized and nonbacterized rhizospheres, certain fungal species were exclusively promoted or specifically suppressed from Pseudomonas-treated soils . In general, fungal diversity and equitability tended to decrease with time while species richness slightly increased . Whilst a total of 29 fungal species were isolated from the mungbean rhizosphere, only eight species colonized the root tissues . CONCLUSIONS: Soil inoculation with Ps . fluorescens CHA0 or CHA0/pME3424 altered fungal community structure in mungbean rhizosphere but strain CHA89 failed to produce such effect . SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas fluorescens-mediated alteration in the composition and structure of fungal communities might have acute or lasting effects on ecosystem functioning . Furthermore, the study provides useful data pertinent to characterization of the fate of genetically modified inoculants (e.g . antibiotic-overproducing Pseudomonas strains) released into the environment.

Mol Ecol, 2003 Nov, 12(11), 3109 - 21
Genes encoding a cellulosic polymer contribute toward the ecological success of Pseudomonas fluorescens SBW25 on plant surfaces; Gal M et al.; Pseudomonas fluorescens SBW25 is a Gram-negative bacterium that grows in close association with plants . In common with a broad range of functionally similar bacteria it plays an important role in the turnover of organic matter and certain isolates can promote plant growth . Despite its environmental significance, the causes of its ecological success are poorly understood . Here we describe the development and application of a simple promoter trapping strategy (IVET) to identify P . fluorescens SBW25 genes showing elevated levels of expression in the sugar beet rhizosphere . A total of 25 rhizosphere-induced (rhi) fusions are reported with predicted roles in nutrient acquisition, stress responses, biosynthesis of phytohormones and antibiotics . One rhi fusion is to wss, an operon encoding an acetylated cellulose polymer . A mutant carrying a defective wss locus was competitively compromised (relative to the wild type) in the rhizosphere and in the phyllosphere, but not in bulk soil . The rhizosphere-induced wss locus therefore contributes to the ecological performance of SBW25 in the plant environment and supports our conjecture that genes inactive in the laboratory environment, but active in the wild, are likely to be determinants of fitness in natural environments.

Mol Ecol, 2003 Nov, 12(11), 3097 - 107
Population dynamics and gene transfer in genetically modified bacteria in a model microcosm; Lilley AK et al.; The horizontal transfer and effects on host fitness of a neutral gene cassette inserted into three different genomic loci of a plant-colonizing pseudomonad was assessed in a model ecosystem . The KX reporter cassette (kanamycin resistance, aph, and catechol 2, 3, dioxygenase, xylE) was introduced on the disarmed transposon mini-Tn5 into: (I) the chromosome of a spontaneous rifampicin resistant mutant Pseudomonas fluorescens SBW25R; (II) the chromosome of SBW25R in the presence of a naturally occurring lysogenic-phage (phage Phi101); and (III) a naturally occurring plasmid pQBR11 (330 kbp, tra+, Hgr) introduced into SBW25R . These bacteria were applied to Stellaria media (chickweed) plants as seed dressings {c . 5 x 104 colony-forming units (cfu)/seed} and the seedlings planted in 16 microcosm chambers containing model plant and animal communities . Gene transfer to pseudomonads in the phyllosphere and rhizosphere was found only in the plasmid treatment (III) . Bacteria in the phage treatment (II) initially declined in density and free phage was detected, but populations partly recovered as the plants matured . Surprisingly, bacteria in the chromosome insertion treatment (I) consistently achieved higher population densities than the unmanipulated control and other treatments . Plasmids were acquired from indigenous bacterial populations in the control and chromosome insertion treatments . Plasmid acquisition, plasmid transfer from inocula and selection for plasmid carrying inocula coincided with plant maturation.

Mol Microbiol, 2003 Nov, 50(4), 1361 - 79
RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0; Valverde C et al.; In the plant-beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two-component system upregulates the production of biocontrol factors, i.e . antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non-exponential growth . When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA-binding protein RsmA, a translational regulator of genes involved in biocontrol . The gene for a second GacA-regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0 . RsmY, like RsmZ, contains several characteristic GGA motifs . The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis . Transcription of rsmY was enhanced by the addition of the strain's own supernatant extract containing a quorum-sensing signal and was abolished in gacS or gacA mutants . An rsmA mutation led to reduced rsmY expression, via a gacA-independent mechanism . Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants . Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4-diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber-Pythium ultimum microcosm . Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs . In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA-mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.

Microbiol Immunol, 2003, 47(10), 709 - 15
Two different mechanisms are involved in the extremely high-level benzalkonium chloride resistance of a Pseudomonas fluorescens strain; Nagai K et al.; A Pseudomonas fluorescens strain, PFRB, which we previously isolated as a contaminant in a batch of benzalkonium chloride (BAC) stock solution, exhibits high-level resistance, not only to BAC, but also to other cationic surfactants belonging to disinfectants classified as quaternary ammonium compounds (QACs) . In this study, we analyzed the resistance mechanism of the strain to BAC and other disinfectants . We obtained results suggesting that two different mechanisms, reduced adsorption of BAC to the cell surface and an energy-dependent mechanism which is most probably an efflux system, were implicated in the high-level resistance to BAC . Reduced adsorption of BAC is likely due to the decreased negative cell surface charge of the strain . The putative efflux system seems to be unique in that it excretes only a certain range of cationic membrane-acting disinfectants belonging to QACs.

Mol Plant Microbe Interact, 2003 Nov, 16(11), 983 - 93
Interactions in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f . sp . radicis-lycopersici; Bolwerk A et al.; The fungus Fusarium oxysporum f . sp . radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P . chlororaphis PCL1391 . Induced systemic resistance is thought to be involved in biocontrol by P . fluorescens WCS365 . The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P . chlororaphis PCL1391 . To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers . Tomato seedlings were inoculated with biocontrol bacteria and planted in an F . oxysporum f . sp . radicis-lycopersici-infested gnotobiotic sand system . Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P . chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.

Can J Microbiol, 2003 Jun, 49(6), 383 - 9
Survival and colonization of rhizobacteria in a tomato transplant system; Yan Z et al.; Plant-growth-promoting rhizobacteria (PGPR) are used on crops most often as seed treatments; however, an alternative application method for transplanted vegetables is mixing PGPR into the soilless medium in which the transplants are grown . Studies were undertaken to compare root colonization and persistence of rifampicin-resistant mutants of PGPR strains Bacillus pumilus SE34 and Pseudomonas fluorescens 89B61, SE34r and 89B61r, on tomato as a function of application method . When the bacteria were incorporated into Promix soilless medium at log 6, 7, and 8 colony- forming units/g, populations of strain SE34r per gram of medium maintained the initial inoculum densities, while populations of 89B61r decreased approximately one to two orders of magnitude by 4 weeks after planting . The populations of each PGPR strain colonizing roots after application into the soilless medium showed a similar pattern at 6 weeks as that at 4 weeks after planting, with higher populations on the whole roots and lateral roots than on the taproots . Strain SE34r but not 89B61r moved upwards and colonized the phyllosphere when incorporated into the soilless medium . Following application as seed treatment, populations of SE34r were significantly higher on upper roots and on the taproot than were populations following application through the soilless medium . Conversely, populations were higher on lower roots and lateral roots following application through the soilless medium than were populations following application as seed treatment . While strain SE34 enhanced plant growth with application both to the medium and as seed treatment, the level of growth promotion was significantly greater with application in the soilless medium . The results indicate that PGPR can be successfully incorporated into soilless media in vegetable transplant production systems.

J Inorg Biochem, 2003 Dec 1, 97(4), 384 - 7
Fluorescent complex of pyoverdin with aluminum; del Olmo A et al.; When a pyoverdin (PV), (a siderophore) from Pseudomonas fluorescens, binds aluminum 1:1, its natural fluorescence almost doubles, whereas PV-Fe is non-fluorescent . Complex formation allows {Al} determination down to 1 mug/l . Fe(III) in the sample interferes with {Al} determination, but added after PV, improves the assay's performance . Ascorbic acid does not eliminate Fe(III) interference . PV-Al fluorescence could have analytical and toxicological applications.

J Enzyme Inhib Med Chem, 2003 Aug, 18(4), 297 - 301
Kinetic studies on the inhibition of GABA-T by gamma-vinyl GABA and taurine; Sulaiman SA et al.; Gamma-aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of gamma-aminobutyric acid . The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: gamma-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid) . A kinetic model that describes the transamination process is proposed . GABA-T from Pseudomonas fluorescens is inhibited by gamma-vinyl GABA and taurine at concentrations of 51.0 and 78.5 mM . Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (Ki) values for gamma-vinyl GABA and taurine were found to be 26 +/- 3 mM and 68 +/- 7 mM respectively . The transamination process of alpha-ketoglutarate was not affected by the presence of gamma-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when alpha-ketoglutarate was the substrate . The inhibition dissociation constant (Kii) for this system was found to be 96 +/- 10 mM . The Michaelis-Menten constant (Km) in the absence of inhibition, was found to be 0.79 +/- 0.11 mM, and 0.47 +/- 0.10 mM for GABA and alpha-ketoglutarate respectively.

Naturwissenschaften, 2003 Oct, 90(10), 468 - 72 Epub 2003 Sep 24.
Biological activities of an extract from Cleome viscosa L . (Capparaceae); Williams LA et al.; Electron micrograph examination of the leaf and stem surfaces of Cleome viscosa L (Family Capparaceae) revealed the presence of secretory glandular trichomes with club-cylinder and cylinder morphologies . In the present study, the leaves and stems of C . viscosa were extracted with hexane and the extract was evaluated for the following biological activities: anti-bacterial, anti-fungal, contact insecticidal and nematicidal . The extract was found to be a potent anti-bacterial agent according to the thin layer chromatography autobiographic assay . Activity-directed isolation studies of the anti-bacterially active compounds led to a 14-member ring cembranoid diterpene being identified as one of the effective agents . Minimum inhibitory concentration (MIC) values (microg/spot) of 5.0 microg/spot and 1.0 microg/spot were found for the diterpene on Bacillus subtilis (Gram-positive) and Pseudomonas fluorescens (Gram-negative), respectively . The diterpene did not inhibit the growth of the fungus Cladosporium cucumerinum . The extract demonstrated a pyrethroid type of contact insecticidal activity on adult Cylas formicarius elegantulus Summer (Coleoptera: Curculionidae) . The extract also had high nematicidal activity with a percentage Abbott's value of 72.69 on the plant parasitic nematode Meloidogyne incognita Chitwood; however, the extract lost its potency upon subfractionation.

Mol Plant Microbe Interact, 2003 Oct, 16(10), 851 - 8
Induced systemic resistance in Arabidopsis thaliana in response to root inoculation with Pseudomonas fluorescens CHA0; Iavicoli A et al.; Root inoculation of Arabidopsis thaliana ecotype Columbia with Pseudomonas fluorescens CHA0r partially protected leaves from the oomycete Peronospora parasitica . The molecular determinants of Pseudomonas fluorescens CHA0r for this induced systemic resistance (ISR) were investigated, using mutants derived from strain CHA0: CHA400 (pyoverdine deficient), CHA805 (exoprotease deficient), CHA77 (HCN deficient), CHA660 (pyoluteorin deficient), CHA631 (2,4-diacetylphloroglucinol {DAPG} deficient), and CHA89 (HCN, DAPG- and pyoluteorin deficient) . Only mutations interfering with DAPG production led to a significant decrease in ISR to Peronospora parasitica . Thus, DAPG production in Pseudomonas fluorescens is required for the induction of ISR to Peronospora parasitica . DAPG is known for its antibiotic activity; however, our data indicate that one action of DAPG could be due to an effect on the physiology of the plant . DAPG at 10 to 100 microM applied to roots of Arabidopsis mimicked the ISR effect . CHA0r-mediated ISR was also tested in various Arabidopsis mutants and transgenic plants: NahG (transgenic line degrading salicylic acid {SA}), sid2-1 (nonproducing SA), npr1-1 (non-expressing NPR1 protein), jar1-1 (insensitive to jasmonic acid and methyl jasmonic acid), ein2-1 (insensitive to ethylene), etr1-1 (insensitive to ethylene), eir1-1 (insensitive to ethylene in roots), and pad2-1 (phytoalexin deficient) . Only jar1-1, eir1-1, and npr1-1 mutants were unable to undergo ISR . Sensitivity to jasmonic acid and functional NPR1 and EIR1 proteins were required for full expression of CHA0r-mediated ISR . The requirements for ISR observed in this study in Peronospora parasitica induced by Pseudomonas fluorescens CHA0r only partially overlap with those published so far for Peronospora parasitica, indicating a great degree of flexibility in the molecular processes leading to ISR.

Appl Microbiol Biotechnol, 2003 Dec, 63(3), 300 - 6 Epub 2003 Oct 11.
A delta-endotoxin encoded in Pseudomonas fluorescens displays a high degree of insecticidal activity; Peng R et al.; The short field-life of Bacillus thuringiensis (Bt) insecticidal crystal protein has limited its use . When the Bt toxin is produced in Pseudomonas fluorescens it can be encapsulated and retain its effectiveness for two to three times longer than other Bt formulations . In order to improve Bt expression, we have synthesized cryIA(c) Bt delta-endotoxin encoding region (GenBank AF537267) according to the usage codon of P . fluorescens and transformed the Bt toxin expression cassette into P . fluorescens strains . T7 RNA polymerase and the T7 promoter system were used to control expression of Bt toxin . SDS-PAGE and Western blotting assay revealed that the delta-endotoxin was expressed as 8% of the total protein in P . fluorescens . In in vitro tests, release of toxin from dead bacteria was demonstrated . Supplementation of diets with Bt toxin-containing Pseudomonas bacterium resulted in high mortality of cabbage butterfly ( Pieris brassicae) larvae.

Microbiology, 2003 Oct, 149(Pt 10), 2909 - 18
Identification of Pseudomonas proteins coordinately induced by acidic amino acids and their amides: a two-dimensional electrophoresis study; Sonawane A et al.; The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) are excellent growth substrates for many pseudomonads . This paper presents proteomics data indicating that growth of Pseudomonas fluorescens ATCC 13525 and Pseudomonas putida KT2440 on these amino acids as sole source of carbon and nitrogen leads to the induction of a defined set of proteins . Using mass spectrometry and N-terminal sequencing, a number of these proteins were identified as enzymes and transporters involved in amino acid uptake and metabolism . Most of them depended on the alternative sigma factor sigma(54) for expression and were subject to strong carbon catabolite repression by glucose and citrate cycle intermediates . For a subset of the identified proteins, the observed regulatory effects were independently confirmed by RT-PCR . The authors propose that the respective genes (together with others still to be identified) make up a regulon that mediates uptake and utilization of the abovementioned amino acids.

Microbiol Res, 2003, 158(3), 203 - 13
Role of salicylic acid in systemic resistance induced by Pseudomonas fluorescens against Fusarium oxysporum f . sp . ciceri in chickpea; Saikia R et al.; Selected isolates of Pseudomonas fluorescens (Pf1-94, Pf4-92, Pf12-94, Pf151-94 and Pf179-94) and chemical resistance inducers (salicylic acid, acetylsalicylic acid, DL-norvaline, indole-3-carbinol and lichenan) were examined for growth promotion and induced systemic resistance against Fusarium wilt of chickpea . A marked increase in shoot and root length was observed in P . fluorescens treated plants . The isolates of P . fluorescens systemically induced resistance against Fusarium wilt of chickpea caused by Fusarium . oxysporum f.sp . ciceri (FocRs1), and significantly (P = 0.05) reduced the wilt disease by 26-50% as compared to control . Varied degree of protection against Fusarium wilt was recorded with chemical inducers . The reduction in disease was more pronounced when chemical inducers were applied with P . fluorescens . Among chemical inducers, SA showed the highest protection of chickpea seedlings against wilting . Fifty two- to 64% reduction of wilting was observed in soil treated with isolate Pf4-92 along with chemical inducers . A significant (P = 0.05; r = -0.946) negative correlation was observed in concentration of salicylic acid and mycelial growth of FocRs1 and at a concentration of 2000 microg ml(-1) mycelial growth was completely arrested . Exogenously supplied SA also stimulated systemic resistance against wilt and reduced the disease severity by 23% and 43% in the plants treated with 40 and 80 microg ml(-1) of SA through root application . All the isolates of P . fluorescens produced SA in synthetic medium and in root tissues . HPLC analysis indicated that Pf4-92 produced comparatively more SA than the other isolates . 1700 to 2000 nanog SA g(-1) fresh root was detected from the application site of root after one day of bacterization whereas, the amount of SA at distant site ranged between 400-500 nanog . After three days of bacterization the SA level decreased and was found more or less equal at both the detection sites.

Curr Microbiol, 2003 Aug, 47(2), 153 - 8
Biocontrol of collar rot disease of betelvine (Piper betle L.) caused by Sclerotium rolfsii by using rhizosphere-competent Pseudomonas fluorescens NBRI-N6 and P . fluorescens NBRI-N; Singh A et al.; Collar rot disease of betelvine (Piper betle L.) caused by Sclerotium rolfsii is difficult to control by conventional means by use of chemicals; therefore, use of biocontrol agents is desirable . In the present study, 186 bacterial strains of different morphological types were screened for their biocontrol activity against S . rolfsii under in vitro conditions . Two strains, Pseudomonas fluorescens NBRI-N6 and P . fluorescens NBRI-N, were selected for further studies because of their ability to inhibit the mycelial growth of the pathogen significantly . Spontaneous rifampicin-resistant (Rif) derivatives of P . fluorescens NBRI-N6 and P . fluorescens NBRI-N showing growth rate and membrane protein composition comparable to the wild type were selected to facilitate their monitoring in the rhizosphere . Field trials demonstrated that strain P . fluorescens NBRI-N6 was better than P . fluorescens NBRI-N in increasing the yield of betelvine significantly, whereas a consortium of the two strains controlled the disease more than either of the strains . The screening method should prove useful in identifying rhizosphere bacteria with the greatest potential for controlling diseases caused by phytopathogenic fungi.

Curr Microbiol, 2003 Aug, 47(2), 138 - 43
Indole-3-Acetic acid production in Pseudomonas fluorescens HP72 and its association with suppression of creeping bentgrass brown patch; Suzuki S et al.; Pseudomonas fluorescens HP72, which suppresses the brown patch disease on bentgrass, produces several secondary metabolites, 2,4-diacetylphloroglucinol (2,4-DAPG), HCN, siderophore, and indole-3-acetic acid (IAA) . In this study, IAA biosynthesis in strain HP72 was investigated . After several repeated subcultures, the spontaneous IAA low-producing mutant HP72LI was isolated . The IAA low production of the strain HP72LI was due to the low tryptophan side chain oxidase (TSO) activity . Colonization of strain HP72 on the bentgrass root induced root growth reduction, while strain HP72LI did not induce such growth reduction . The colonization ability of strain HP72 on the bentgrass root is higher than that of strain HP72LI . However, as for biocontrol ability, a significant difference in both strains was not detected . IAA production by strain HP72 may play a role in the construction of short root systems and take advantage of root colonization, but does not contribute to the biocontrol properties of P . fluorescens HP72.

Chemosphere, 2003 Dec, 53(8), 889 - 97
Optimisation of a microbial bioassay for contaminated soil monitoring: bacterial inoculum standardisation and comparison with Microtox assay; Abbondanzi F et al.; This work represents the first step to set up a toxicity testing procedure and to evaluate the sensitivity of the test microorganism to several classes of environmental pollutants . First, three different techniques were employed to standardise the microbial inoculum, then two different toxicity assessment protocols have been compared: Microtox and a dehydrogenase (DHase) activity inhibition test . The main goal was the optimisation of a microbial bioassay based on the dehydrogenase activity (DHase) inhibition in Pseudomonas fluorescens bacterial strain ATCC 13525 . Triphenyl tetrazolium chloride (TTC) was used as electron acceptor and its reduction produces Triphenyl formazane (TPF) . The P . fluorescens DHase inhibition bioassay was investigated for being a reliable and rapid method for assessing toxicity . The optimisation of the operating conditions resulted in a repeatable bioassay . Then, P . fluorescens and Vibrio fischeri sensitivity were firstly compared by testing Zn++, one of the reference compounds for Microtox test . In addition, other compounds (Ni++, Cd++, Cu++, phenol) were also tested with both bioassays . A high statistical significance of data was obtained with the logistic curve . The present work has demonstrated that P . fluorescens is as sensitive as Microtox culture (V . fischeri), for some of the metal ions . With reference to organic compounds, the lower sensitivity of P . fluorescens to phenol makes its use difficult in organic polluted samples.

BMC Biochem . 2003 Sep 24;4(1):13.
Comparative inhibition by substrate analogues 3-methoxy- and 3-hydroxydesaminokynurenine and an improved 3 step purification of recombinant human kynureninase; Walsh HA et al.; BACKGROUND: Kynureninase is a key enzyme on the kynurenine pathway of tryptophan metabolism . One of the end products of the pathway is the neurotoxin quinolinic acid which appears to be responsible for neuronal cell death in a number of important neurological diseases . This makes kynureninase a possible therapeutic target for diseases such as Huntington's, Alzheimer's and AIDS related dementia, and the development of potent inhibitors an important research aim . RESULTS: Two new kynurenine analogues, 3-hydroxydesaminokynurenine and 3-methoxydesaminokynurenine, were synthesised as inhibitors of kynureninase and tested on the tryptophan-induced bacterial enzyme from Pseudomonas fluorescens, the recombinant human enzyme and the rat hepatic enzyme . They were found to be mixed inhibitors of all three enzymes displaying both competitive and non competitive inhibition . The 3-hydroxy derivative gave low Ki values of 5, 40 and 100 nM respectively . An improved 3-step purification scheme for recombinant human kynureninase was also developed . CONCLUSION: For kynureninase from all three species the 2-amino group was found to be crucial for activity whilst the 3-hydroxyl group played a fundamental role in binding at the active site presumably via hydrogen bonding . The potency of the various inhibitors was found to be species specific . The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase . The modified purification described is relatively quick, simple and cost effective.

J Microbiol Methods, 2003 Oct, 55(1), 221 - 9
Whole-cell bacterial sensors for the monitoring of phosphate bioavailability; Dollard MA et al.; A phosphate sensor plasmid was constructed, in which the inducible promoter of the alkaline phosphatase gene (phoA) from Escherichia coli is fused to the bioluminescence genes from Vibrio fischeri . The reporter construct was introduced into E . coli MG1655 and the rhizosphere coloniser Pseudomonas fluorescens DF57, which produced light in a dose-dependent manner when exogenous phosphate concentrations fell below 60 and 40 microM, respectively . These strains also responded to various organic and inorganic phosphorus compounds . Their ability to distinguish the bioavailable portion of phosphate in standard solution was demonstrated using different phosphate ligands . When applying the bioassay to wastewater samples, luminescence patterns correlated with phosphate concentrations determined by standard chemical procedure . These results indicated that phoA::lux-based bacterial sensors may serve as tools for the assessment of phosphate bioavailability.

Nature, 2003 Sep 4, 425(6953), 72 - 4
Evolution of cooperation and conflict in experimental bacterial populations; Rainey PB et al.; A fundamental problem in biology is the evolutionary transition from single cells to multicellular life forms . During this transition the unit of selection shifts from individual cells to groups of cooperating cells . Although there is much theory, there are few empirical studies . Here we describe an evolutionary transition that occurs in experimental populations of Pseudomonas fluorescens propagated in a spatially heterogeneous environment . Cooperating groups are formed by over-production of an adhesive polymer, which causes the interests of individuals to align with those of the group . The costs and benefits of cooperation, plus evolutionary susceptibility to defecting genotypes, were analysed to determine conformation to theory . Cooperation was costly to individuals, but beneficial to the group . Defecting genotypes evolved in populations founded by the cooperating type and were fitter in the presence of this type than in its absence . In the short term, defectors sabotaged the viability of the group; but these findings nevertheless show that transitions to higher orders of complexity are readily achievable, provide insights into the selective conditions, and facilitate experimental analysis of the evolution of individuality.

Environ Sci Technol, 2003 Aug 15, 37(16), 3555 - 9
Fate of uranyl in a quaternary system composed of uranyl, citrate, goethite, and Pseudomonas fluorescens; Bencheikh-Latmani R et al.; This study investigated the partitioning of uranyl within a quaternary system made up of uranyl, citrate, goethite, and the bacterium Pseudomonas fluorescens . In the absence of cells, uranyl was sorbed to goethite as a complex involving surface groups and/or citrate . Measurements of the evolution of CO2 indicated that the addition of bacterial cells lead to the gradual biodegradation of citrate . Throughout the biodegradation process, uranyl remained sorbed to the insoluble fraction comprised of goethite and cells . EXAFS (Extended X-ray Absorption Fine Structure) measurements showed that bacterial cells outcompeted goethite for uranyl under the experimental conditions and caused the repartitioning of uranyl from goethite to cell matter, independently from citrate degradation . Citrate degradation caused further release of uranyl from goethite surfaces, followed by subsequent association of uranyl with cells . At long equilibration times (3 months), cell lysis and phosphate release resulted in the precipitation of an autunite-like phase . This work suggests that bacterial degradation of uranyl-complexing ligands in contaminated subsurface media containing iron oxides should not necessarily lead to an increase in the mobility of uranyl.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003 Sep, 38(9), 1857 - 66
Bioremediation of crude oil contaminated soil by bioaugmentation of Pseudomonas fluorescens NS1; Barathi S et al.; A feasibility study was conducted to evaluate the efficiency of Pseudomonas fluorescens NS1 bioaugmented to stimulate in situ bioremediation of crude oil-contaminated soil with different amendments in treatment units . Pure culture of P . fluorescens NS1 was isolated from a petroleum-contaminated soil . The rate of degradation of petroleum hydrocarbons by the indigenous soil microflora and in the presence of P . fluorescens NS1 was assessed with the addition of nutrients and bulking agents for a period of about 35 days . The study showed that addition of wheat bran as bulking agent rapidly enhanced bioremediation of crude oil-contaminated soil compared to amendments in other treatment units.

Org Biomol Chem, 2003 Jan 21, 1(2), 288 - 95
Differential effects of bromination on substrates and inhibitors of kynureninase from Pseudomonas fluorescens; Heiss C et al.; A series of brominated compounds has been synthesized and evaluated as substrates and inhibitors of kynureninase from Pseudomonas fluorescens . Both 3-bromo-L-kynurenine and 5-bromo-L-kynurenine were found to be substrates with similar k(cat) values to L-kynurenine, but the K(m) value for 3-bromo-L-kynurenine is very high (ca . 2 mM) compared to that for 5-bromo-L-kynurenine (11 microM) and L-kynurenine (25 microM) . Both isomers of bromokynurenine react with kynureninase within the dead time of the stopped-flow instrument (ca . 1 ms) to form quinonoid intermediates with a lambda max of 494 nm that decay with rate constants of 300-600 s-1, similar to L-kynurenine . The two diastereomers of 5-bromodihydro-L-kynurenine were also prepared, and are more potent inhibitors than dihydro-L-kynurenines . (4R)-5-Bromodihydro-L-kynurenine is one of the most potent inhibitors of P . fluorescens kynureninase found to date (Ki = 55 nM) and also acts as a slow substrate; the (4S)-epimer, on the other hand, shows no measurable substrate activity, but it is a potent competitive inhibitor with a Ki value of 170 nM . In contrast, brominated analogs of (S)-(2-aminophenyl)-L-cysteine S,S-dioxide, (S)-(2-amino-4-bromophenyl)-L-cysteine S,S-dioxide and (S)-(2-amino-5-bromophenyl)-L-cysteine S,S-dioxide are competitive inhibitors of kynureninase, with Ki values of about 300 and 400 nm, respectively, about ten-fold higher than the value of 27 nM obtained for the parent compound . These results suggest that the binding modes of substrates and the various classes of inhibitors in the active site of kynureninase are different.

Clin Chim Acta, 2003 Sep, 335(1-2), 9 - 20
Advances in clinical laboratory tests for inflammatory bowel disease; Nakamura RM et al.; Inflammatory bowel disease (IBD) is a generic term that refers to Crohn's disease and chronic ulcerative colitis (UC) . The CD and UC are considered to be distinct forms of IBD; but there is a subgroup of CD with a UC-like presentation . The genetic factors play a significant role in IBD . IBD is associated with a strong familial pattern . Recent studies support the hypothesis that IBD patients have a dysregulated immune response to endogenous bacteria in the gastrointestinal tract . The serologic responses seen in Crohn's disease include antibodies to Saccharomyces cerevisiae, mycobacteria, bacteroides and E . coli . The pANCA antibody seen in UC and CD has been demonstrated to react with epitopes of H1 histone, Bacteroides caccae (Ton-B linked outer membrane protein), Pseudomonas fluorescens-associated bacterial protein I-2, mycobacterial histone 1 homologue called Hup B . In recent years, several serologic markers have been found to be useful for the diagnosis and differentiation of CD and UC . These markers include the following antibodies: (a) pANCA, (b) ASCA, (c) anti-pancreatic antibody, (d) OmpC antibody and (e) I-2 antibody and antibodies to anaerobic coccoid rods . The application of a panel of markers with the use of an algorithm (i.e . IBD First Step) can identify specific subtypes of IBD that have different clinical courses and progression of the diseases . The serologic markers are useful for the diagnosis and management of CD and UC patients.

Microb Pathog, 2003 Sep, 35(3), 95 - 106
Cytotoxic effects of the lipopolysaccharide from Pseudomonas fluorescens on neurons and glial cells; Picot L et al.; Pseudomonas fluorescens is an emerging pathogen closely related to Pseudomonas aeruginosa . In the present study, the effect of the lipopolysaccharide (LPS) from P . fluorescens MF37 was investigated using indicators of apoptosis and necrosis and was compared to the effect of the LPS from P . aeruginosa PAO1 . Capillary electrophoresis analysis of the LPS from P . fluorescens MF37 revealed the existence of three forms of the endotoxin and the absence of homology with the LPS from P . aeruginosa . In neurons and glial cells the LPS from P . fluorescens induced major morphological changes including a condensation of the cytoplasmic proteins, a leakage of the cytoplasmic content, the formation of blebs on the nuclear membrane and a marked reorganization of the cytoskeleton . In glial cells, the LPS from P . fluorescens provoked the migration of phosphatidylserine at the surface of the cytoplasmic membrane, a sign of apoptosis, but this reaction was associated to an increase in the permeability to propidium iodide characteristic of necrosis . Biochemical studies revealed an important activation of an inducible nitric oxide synthase and a release of lactate dehydrogenase, a stable cytosolic enzyme . These results demonstrate that the LPS from P . fluorescens induces apoptosis and a concomitant and limited necrosis, reveal the unexpected cytotoxicity of this endotoxin and provide the first demonstration of the apoptotic effect of a non-aeruginosa Pseudomonas on nerve cells.

Brain Res, 2003 Sep 5, 983(1-2), 185 - 92
Pseudomonas fluorescens lipopolysaccharide inhibits both delayed rectifier and transient A-type K+ channels of cultured rat cerebellar granule neurons; Mezghani-Abdelmoula S et al.; Pseudomonas fluorescens is a Gram-negative bacillus closely related to the pathogen P . aeruginosa known to provoke infectious disorders in the central nervous system (CNS) . The endotoxin lipopolysaccharide (LPS) expressed by the bacteria is the first infectious factor that can interact with the plasma membrane of host cells . In the present study, LPS extracted from P . fluorescens MF37 was examined for its actions on delayed rectifier and A-type K(+) channels, two of the main types of voltage-activated K(+) channels involved in the action potential firing . Current recordings were performed in cultured rat cerebellar granule neurons at days 7 or 8, using the whole-cell patch-clamp technique . A 3-h incubation with LPS (200 ng/ml) markedly depressed both the delayed rectifier (I(KV)) and transient A-type (I(A)) K(+) currents evoked by depolarizations above 0 and -40 mV, respectively . The percent decrease of I(KV) and I(A) ( approximately 30%) did not vary with membrane potential, suggesting that inhibition of both types of K(+) channels by LPS was voltage-insensitive . The endotoxin did neither modify the steady-state voltage-dependent activation properties of I(KV) and I(A) nor the steady-state inactivation of I(A) . The present results suggest that, by inhibiting I(KV) and I(A), LPS applied extracellulary increases the action potential firing in cerebellar granule neurons . It is concluded that P . fluorescens MF37 may provoke in the CNS disorders associated with sever alterations of membrane ionic channel functions.

Dis Aquat Organ, 2003 Jul 8, 55(2), 117 - 23
Molecular cloning, expression of orange-spotted grouper goose-type lysozyme cDNA, and lytic activity of its recombinant protein; Yin ZX et al.; Lysozyme acts as a non-specific innate-immunity molecule against the invasion of bacteria pathogens . A leukocyte cDNA library of orange-spotted grouper Epinephelus coioides was constructed and the goose-type (g-type) lysozyme cDNA was isolated . The complete cDNA consists of an open reading frame of 585 bp encoding a protein of 194 amino acids . This protein shows a 72.2% amino acid sequence identity with the flounder g-type lysozyme . Similar to most other species, the glu catalytic residue in g-type lysozymes of the grouper is conserved . Furthermore, like the flounder and carp, the 4 conserved cysteine residues identified in avian and mammalian g-type lysozymes were also absent from the grouper . Northern blot analysis indicated that the g-type lysozyme was expressed in intestine, liver, spleen, anterior kidney, posterior kidney, heart, gill, muscle and leukocytes . In addition, RT-PCR analysis detected the g-type lysozyme transcripts in the stomach, brain and ovary . When an orange-spotted grouper was injected with Vibrio alginolyticus, the number of lysozyme mRNA transcripts detected in the stomach, spleen, anterior kidney, posterior kidney, heart, brain and leucocytes increased 72 h after injection . Recombinant grouper g-type lysozyme produced in the Escherichia coli expression system showed lytic activity against Micrococcus lysodeikticus, V . alginolyticus from Epinephelus fario, V . vulnificus from culture water, Aeromonas hydrophila from soft-shell turtle, A . hydrophila from goldfish and V . parahaemolyticus, Pseudomonas fluorescens and V . fluvialis from culture water.

J Med Microbiol, 2003 Sep, 52(Pt 9), 759 - 63
Development of a diagnostic PCR assay that targets a heat-shock protein gene (groES) for detection of Pseudomonas spp . in cystic fibrosis patients; Clarke L et al.; Laboratory detection of Pseudomonas spp., in particular Pseudomonas aeruginosa, remains an important assay in the management of patients with cystic fibrosis (CF) . As the groES and groEL genes of P . aeruginosa have now been cloned and their nucleotide sequences determined, the aim of this study was to develop a novel PCR assay for the detection of Pseudomonas spp . from patients with CF by employing conserved primer regions of the groE heat-shock protein domain gene . A PCR assay was designed that targeted a 536 bp region of the groE gene to detect Pseudomonas spp . PCR amplification of genomic DNA from extracted organisms generated an amplicon of the expected size (approx . 536 bp) for all P . aeruginosa (n = 60), Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas stutzeri isolates examined, but did not produce a positive amplicon for several other genera and species that are commonly isolated from the sputum of CF patients . RFLP analysis of the amplicons of all P . aeruginosa isolates demonstrated a single RFLP type that consisted of three bands at approximately 80, 190 and 250 bp; direct sequencing of the amplicons demonstrated the presence of a single sequence type, indicating the highly conserved nature of this region . In addition, the assay successfully produced a positive signal from primary non-selective plates of three known P . aeruginosa culture-positive CF patients, but was unable to generate a signal in a further six CF patients who had no history of infection with P . aeruginosa or other Pseudomonas spp . This assay is recommended to detect the presence of Pseudomonas spp., including P . aeruginosa, from primary culture plates that originate from laboratory analysis of CF patients' sputum, particularly at review, in those patients with no previous history of Pseudomonas infection or those who appear to be transiently colonized by this organism . Employment of such molecular methodologies, in conjunction with routine clinical sputum cultures, may provide improved information on the microbial status of CF patients, which will aid clinicians in both optimum patient management in terms of antibiotic regimes and CF centre infection-control practices.

Proc R Soc Lond B Biol Sci, 2003 Aug 7, 270(1524), 1645 - 50
Divergent evolution during an experimental adaptive radiation; MacLean RC et al.; How repeatable a process is evolution? Comparative studies of multicellular eukaryotes and experimental studies with unicellular prokaryotes document the repeated evolution of adaptive phenotypes during similar adaptive radiations, suggesting that the outcome of adaptive radiation is broadly reproducible . The goal of this study was to test this hypothesis by using phenotypic traits to infer the genetic basis of adaptation to simple carbon-limited environments in an extensive adaptive radiation . We used a clone of the bacterium Pseudomonas fluorescens to found two sets of experimental lines . The first set of lines was allowed to adapt to one of 23 novel environments for 1100 generations while the second set of lines was allowed to accumulate mutations by drift for 2000 generations . All lines were then assayed in the 95 environments provided by Biolog microplates to determine the phenotypic consequences of selection and drift . Replicate selection lines propagated in a common environment evolved similar adaptive components of their phenotype but showed extensive variation in non-adaptive phenotypic traits . This variation in non-adaptive phenotypic traits primarily resulted from the ascendance of different beneficial mutations in different lines . We argue that these results reconcile experimental and comparative approaches to studying adaptation by demonstrating that the convergent phenotypic evolution that occurs during adaptive radiation may be associated with radically different sets of beneficial mutations.

Microbiol Res, 2003, 158(2), 163 - 8
Solubilization of inorganic phosphate and plant growth promotion by cold tolerant mutants of Pseudomonasfluorescens; Katiyar V et al.; A study for screening and selection of cold tolerant mutants of Pseudomonas fluorescens strains GRS1, PRS9 and ATCC13525 based on 'P' solubilization ability and subsequent effect on plant growth promotion under in vitro and in situ conditions was conducted . Of all the mutants tested, two were selected, as there was a 21-fold increase in CRPF, (GRS, mutant) and a 10-fold decrease in CRPF7 (PRS9 mutant) over their respective wild types . Under in vitro conditions at 10 degrees C, these cold tolerant mutants exhibited increased plant growth indicating their functionality at low temperature . Subsequently, greenhouse trials using soil-plant microcosms were conducted which revealed that CRPF, (high 'P' solubilizer) was a good rhizosphere colonizer showing a significant increase in root (30 and 20%) and shoot length (20 and 24%) of mung bean, both in sterilized and unsterilized soil, respectively . On the contrary, CRPF, (low 'P' solubilizer) did not stimulate plant growth . Furthermore, sand experiments indicated that tricalcium phosphate served as better phosphorus source for CRPF2 treated mung bean seeds.

Biodegradation, 2003 Apr, 14(2), 65 - 72
Heteroaromatic monothiocarboxylic acids from Pseudomonas spp; Budzikiewicz H; Pyridine derivatives substituted with monothiocarboxylic acid groups are the unique metabolites of certain Pseudomonas species . Pyridine-2,6-di-(monothiocarboxylic acid) 1a was found during a screening program for antibiotically active bacterial metabolites due to its ability to complex Fe3+ . The structure of this complex, its redox behavior and the biogenesis of the ligand molecule were studied in detail . This lead to the discovery of a new class of natural products, viz . acylsulfenic acid derivatives . Interest in la was revived shortly when complexes with other metals were studied as models for sulfur-containing enzymes . It could also be shown that a quinoline monothiocarboxylic acid derivative acted as an alternative siderophore for Pseudomonas fluorescens . But a real renaissance was observed only when the role of la in the degradation of CCl4 by Pseudomonas stutzeri became evident.

Acta Trop, 2003 Aug, 87(3), 341 - 3
Field evaluation of a formulation of Pseudomonas fluorescens against Culex quinquefasciatus larvae and pupae; Sadanandane C et al.; VCRC B426, 0.09% emulsifiable concentrate (EC) formulation developed from a metabolite of Pseudomonas fluorescens was tested for efficacy against Culex quinquefasciatus larvae and pupae . At application rates of 100, 200, 300 ml/m2, the formulation caused 100% elimination of larvae and pupae at day 1 after treatments and >80% reduction in pupal density for periods of 7, 12 and 11 days in cesspits and 5, 9 and 10 days in U-shaped drains . In both the habitats, the efficacy of the formulation against pupae was 1.7 times more at 200 ml/m2 than at 100 ml/m2 . An increase in dosage to 300 ml/m2 did not improve the efficacy in cesspits but a marginal increase was observed in drains.

Lett Appl Microbiol, 2003, 37(2), 109 - 14
Non-pathogenic Fusarium solani represses the biosynthesis of nematicidal compounds in vitro and reduces the biocontrol of Meloidogyne javanica by Pseudomonas fluorescens in tomato; Siddiqui IA et al.; AIMS: The aim of the present investigation was to determine the influence of various Fusarium solani strains on the production of nematicidal agent(s) in vitro and biocontrol of Meloidogyne javanica in tomato by Pseudomonas fluorescens strains CHA0 and CHA0/pME3424 . METHODS AND RESULTS: Culture filtrates (CF) of P . fluorescens strain CHA0 and its diacetylphloroglucinol-overproducing derivative CHA0/pME3424 caused substantial mortality of M . javanica juveniles in vitro . Bacterial growth medium amended with the growth medium of F . solani repressed the nematicidal activity of the bacteria . Methanol extract of F . solani CF resulting from Czapek's Dox liquid (CDL) medium without zinc amendment repressed the nematicidal activity of the bacteria while the CF obtained from CDL medium amended with zinc did not . Conidial suspension of F . solani strain Fs5 (repressor strain for the biosynthesis of nematicidal compounds in P . fluorescens) reduced biocontrol potential of the bacterial inoculants against M . javanica in tomato while strain Fs3 (non-repressor) did not . CONCLUSIONS: Fusarium solani strains with increased nematicidal activity repress the biosynthesis of nematicidal compounds by P . fluorescens strains in vitro and greatly alter its biocontrol efficacy against root-knot nematode under natural conditions . SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium solani strains are distributed worldwide and found in almost all the agricultural fields which suggest that some mycotoxin-producing strains will also be found in almost any soil sample taken . Besides the suppressive effect of these metabolite-producing strains on the production of nematicidal compound(s) critical in biocontrol, F . solani strains may also affect the performance of mycotoxin-sensitive biocontrol bacteria effective against plant-parasitic nematodes.

Mol Plant Microbe Interact, 2003 Jul, 16(7), 634 - 44
GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0; Zuber S et al.; In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system . Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes . Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs . Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal . Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities . In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f . sp . radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.

Protein Eng, 2003 May, 16(5), 357 - 64
A systematic approach for yielding a potential pool of enzymes: practical case for chiral resolution of (R,S)-ketoprofen ethyl ester; Kim JY et al.; A systematic approach for the selection of potential biocatalysts from a natural source was developed and then a practical application was addressed . The approach that involves systematically combined conventional screening methods and current tools comprises the following consecutive steps: strain enrichment for activity screening, identification of positive strains, choosing whole genome-sequenced strains as candidates, gathering information about responsible enzymes, bioinformatic analyses and gene mining, probing genetic molecules and then functional expression . The target compound (R,S)-ketoprofen ethyl ester was to be resolved into an enantiomer, and a potential esterase from Pseudomonas fluorescens KCTC 1767 was prepared by the proposed procedure . The enzyme had a high activity and also strict selectivity for the enantiomer (S)-ketoprofen and was suitable therefore as a biocatalyst for practical use . The result achieved by the combined approach could not easily be obtained using other approaches with typical procedures . Hence the approach proposed here should be of considerable use for the screening of potential enzymes, particularly for enzymes with desired activity to unnatural substrates, from conditionally expressed and/or repressed proteins that are distributed widely in natural pools under normal conditions.

Biochem J, 2003 Oct 1, 375(Pt 1), 141 - 9
On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase; Klimacek M et al.; X-ray structure of the Pseudomonas fluorescens mannitol 2-dehydrogenase ternary complex with NAD+ and D-mannitol suggests that Lys-295 provides catalytic base assistance to secondary alcohol group oxidation . We have replaced Lys-295 by site-directed mutagenesis with alanine or methionine and evaluated the catalytic significance of side-chain substitution by kinetic analysis of restoration of activity with external amines, and from pH and solvent isotope effects on the reaction catalysed by K295A (Lys-295-->Ala mutant) . K295A and K295M (Lys-295-->Met mutants) show 3x10(4)- and 2x10(6)-fold lower turnover numbers respectively for D-mannitol oxidation (kcatO) at pH 10.0 than the wild-type . The second-order rate constant for non-covalent rescue of activity (kB) by free methylamine base is 31 M(-1) x s(-1) for K295A, but only 0.021 M(-1) x s(-1) for K295M . A Bronsted relationship of log kB (corrected for molecular size effects) and pKa of the external amine is linear (slope beta=0.66+/-0.16; r2=0.99) for K295A-catalysed D-mannitol oxidation at pH 10.0 . The kcatO values of K295A in H2O and 2H2O are linearly dependent on {OL-} in the pL range 7.5-10.5 (where L is 1H or 2H) . The solvent isotope effect on kcatO is 0.69 . The time course of D-fructose reduction by K295A at pH 8.2 displays a pre-steady-state burst of NADH consumption . These data support a mechanism in which the epsilon -NH2 group of Lys-295 participates in an obligatory pH-dependent, pre-catalytic equilibrium which may control alcohol/alkoxide equilibration of enzyme-bound D-mannitol and activates the C2 atom for subsequent catalytic oxidation by NAD+.

ScientificWorldJournal, 2002 May 31, 2, 1501 - 6
Biodegradation of chlorsulfuron and metsulfuron-methyl by Aspergillus niger; Zanardini E et al.; In this work, investigations were performed under laboratory conditions of the degradation ability by a common soil fungus, Aspergillus niger, toward chlorsulfuron and metsulfuron-methyl . The results were very encouraging (79% for chlorsulfuron and 61% for metsulfuron-methyl of total degradation), especially compared to those registered in our previous studies with a Pseudomonas fluorescens strain B2 (about 21 to 32%) . Furthermore, the chemical degradation of the two compounds was studied and two products (1{2-methoxy-benzene-1-sulfonyl}-7-acetyltriuret and 1{2-chlorobenzene-1-sulfonyl}-7-acetyltriuret) were isolated and characterised by hydrolysis in acidic conditions . Our aim in the future will be the identification of intermediate metabolites by HPLC and LC-MS analyses in order to identify the degradative pathway by the fungal strain and to compare this to those obtained by chemical degradation and by P . fluorescens strain.

Org Lett, 2003 Jun 12, 5(12), 2047 - 50
Benzaldehyde lyase-catalyzed enantioselective carboligation of aromatic aldehydes with mono- and dimethoxy acetaldehyde; Demir AS et al.; {reaction: see text} Benzaldehyde lyase from the Pseudomonas fluorescens catalyzes the reaction of aromatic aldehydes with methoxy and dimethoxy acetaldehyde and furnishes (R)-2-hydroxy-3-methoxy-1-arylpropan-1-one and (R)-2-hydroxy-3,3-dimethoxy-1-arylpropan-1-one in high yields and enantiomeric excess via acyloin linkage . Aromatic aldehydes and benzoins are converted into enamine-carbanion-like intermediates prior to carboligation.

Appl Environ Microbiol, 2003 Jun, 69(6), 3333 - 43
Identification and manipulation of soil properties to improve the biological control performance of phenazine-producing Pseudomonas fluorescens; Ownley BH et al.; Pseudomonas fluorescens 2-79RN(10) protects wheat against take-all disease caused by Gaeumannomyces graminis var . tritici; however, the level of protection in the field varies from site to site . Identification of soil factors that exert the greatest influence on disease suppression is essential to improving biocontrol . In order to assess the relative importance of 28 soil properties on take-all suppression, seeds were treated with strain 2-79RN(10) (which produces phenazine-1-carboxylate {PCA(+)}) or a series of mutants with PCA(+) and PCA(-) phenotypes . Bacterized seeds were planted in 10 soils, representative of the wheat-growing region in the Pacific Northwest . Sixteen soil properties were correlated with disease suppression . Biocontrol activity of PCA(+) strains was positively correlated with ammonium-nitrogen, percent sand, soil pH, sodium (extractable and soluble), sulfate-sulfur, and zinc . In contrast, biocontrol was negatively correlated with cation-exchange capacity (CEC), exchangeable acidity, iron, manganese, percent clay, percent organic matter (OM), percent silt, total carbon, and total nitrogen . Principal component factor analysis of the 16 soil properties identified a three-component solution that accounted for 87 percent of the variance in disease rating (biocontrol) . A model was identified with step-wise regression analysis (R(2) = 0.96; Cp statistic = 6.17) that included six key soil properties: ammonium-nitrogen, CEC, iron, percent silt, soil pH, and zinc . As predicted by our regression model, the biocontrol activity of 2-79RN(10) was improved by amending a soil low in Zn with 50 micro g of zinc-EDTA/g of soil . We then investigated the negative correlation of OM with disease suppression and found that addition of OM (as wheat straw) at rates typical of high-OM soils significantly reduced biocontrol activity of 2-79RN(10).

Curr Microbiol, 2003 Jul, 47(1), 65 - 70
Enzymatic dehalogenation of pentachlorophenol by Pseudomonas fluorescens of the microbial community from tannery effluent; Shah S et al.; Four different bacterial isolates obtained from a stable bacterial consortium were capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source . The consortium was developed by continuous enrichment in the chemostat . The degradation of PCP by bacterial strain was preceded through an oxidative route as indicated by accumulation of tetrachloro-rho-hydroquinone and dichlorohydroquinone as determined by high performance liquid chromatography (HPLC) . Among the four isolates, Pseudomonas fluorescens exhibited maximum degradation capability and enzyme production . PCP-monooxygenase enzyme was extracted from culture extract and fractionated by DEAE-cellulose ion exchange chromatography . The molecular weight of the enzyme, purified from Pseudomonas fluorescens, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography was found to be 24000 Da.

Curr Microbiol, 2003 Jul, 47(1), 32 - 9
The metabolism of aluminum citrate and biosynthesis of oxalic acid in Pseudomonas fluorescens; Appanna VD et al.; (13)CNMR and (1)HNMR studies revealed that aluminum citrate (Al-citrate) was metabolized intracellularly and that oxalic acid was an important product in the Al-stressed cells . This dicarboxylic acid was produced via the oxidation of glyoxylate, a precursor generated through the cleavage of isocitrate . In the control cells, citrate was biotransformed essentially with the aid of regular tricarboxylic cycle (TCA) enzymes . However, these control cells were able neither to uptake nor to metabolize Al-citrate . Al-stressed cells obtained at 38-40 h of growth showed maximal Al-citrate uptake and biotransforming activities . At least a fourfold increase in the activity of the enzyme isocitrate lyase (ICL, E . C . 4.1.3.1) has been observed in the Al-stressed cells compared with the control cells . The transport of Al-citrate was sensitive to p-dinitrophenol and sodium azide, but not to dicyclohexylcarbodiimide . Experiments with the dye 9-aminoacridine revealed that the translocation of Al-citrate led to an increase in intracellular pH . Thus, it appears that after the uptake of Al-citrate, this complex is metabolized intracellularly.

J Bacteriol, 2003 Jun, 185(12), 3515 - 23
The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation; Gimmestad M et al.; Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid . Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG . All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii . An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end . The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG . A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues . This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.

Chem Biol, 2003 May, 10(5), 419 - 30
Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586; El-Sayed AK et al.; The polyketide antibiotic mupirocin (pseudomonic acid) produced by Pseudomonas fluorescens NCIMB 10586 competitively inhibits bacterial isoleucyl-tRNA synthase and is useful in controlling Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus . The 74 kb mupirocin biosynthesis cluster has been sequenced, and putative enzymatic functions of many of the open reading frames (ORFs) have been identified . The mupirocin cluster is a combination of six larger ORFs (mmpA-F), containing several domains resembling the multifunctional proteins of polyketide synthase and fatty acid synthase type I systems, and individual genes (mupA-X and macpA-E), some of which show similarity to type II systems (mupB, mupD, mupG, and mupS) . Gene knockout experiments demonstrated the importance of regions in mupirocin production, and complementation of the disrupted gene confirmed that the phenotypes were not due to polar effects . A model for mupirocin biosynthesis is presented based on the sequence and biochemical evidence.

Water Res, 2003 Jun, 37(11), 2618 - 26
Removal of fish pathogenic bacteria in biological sand filters; Bomo AM et al.; Documentation is required to evaluate the use of infiltration systems as an alternative method for removal of fish pathogenic bacteria in wastewater from fish-farms . This study was performed to investigate the removal of bacterial fish pathogens in biological sand filters . A second aim of the study was to evaluate the bacteria used in the study in order to find a suitable model organism for future experiments . Low-strength wastewater from an inland freshwater salmonid farm was intermittently loaded (70 mm/day in 24 doses) to filter columns containing either fine sand (d(10)=0.25) or coarse sand (d(10)=0.86) . After a wastewater loading period of 10 weeks, separate sand columns were seeded with Yersinia ruckeri, Pseudomonas fluorescens, Aeromonas hydrophila and Aeromonas salmonicida subsp . salmonicida, respectively, for a period of 30 days . All the bacteria showed the same removal performance during the experiment, with a significantly lower removal in the beginning of the experiment (day 1-7) compared to mid- and late-phase (day 12-30) . In mid- and late-phase the removal stabilized at a high level (>99.9%) for all the bacteria . The hydrophobic cell surface properties of the Aeromonads were higher than Ps . fluorescens and Y . ruckeri . This can possibly explain the significantly higher (P<0.05) removal efficiencies seen for A . hydrophila and A . salmonicida subsp . salmonicida compared to Y . ruckeri and Ps . fluorescens . Results were promising with regard to the use of low-cost infiltration systems as an alternative disinfection method for fish-farm wastewater . Following the criteria for a suitable model organism (removal efficiency, detection in filter effluent and die-off in storage tanks), Y . ruckeri was found to be a feasible model organism for use in future experiments.

Mikrobiologiia, 2003 Mar-Apr, 72(2), 194 - 8
{Fluorene cometabolism by Rhodococcus rhodochrous and Pseudomonas fluorescens}; Baboshin MA et al.; The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase . Under certain cultivation conditions, fluorene was completely consumed from the medium . The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source . An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.

Chemistry, 2003 May 9, 9(9), 1933 - 9
Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3bromo-2-methylpropanoate and ethyl 3phenylbutyrate; Horsman GP et al.; Directed evolution combined with saturation mutagenesis identified six different point mutations that each moderately increases the enantioselectivity of an esterase from Pseudomonas fluorescens (PFE) towards either of two chiral synthons . Directed evolution identified a Thr230Ile mutation that increased the enantioselectivity from 12 to 19 towards methyl (S)-3-bromo-2-methylpropanoate . Saturation mutagenesis at Thr230 identified another mutant, Thr230Pro, with higher-than-wild-type enantioselectivity (E=17) . Previous directed evolution identified mutants Asp158Asn and Leu181Gln that increased the enantioselectivity from 3.5 to 5.8 and 6.6, respectively, towards ethyl (R)-3-phenylbutyrate . In this work, saturation mutagenesis identified other mutations that further increase the enantioselectivity to 12 (Asp158Leu) and 10 (Leu181Ser) . A homology model of PFE indicates that all mutations lie outside the active site, 12-14 A from the substrate and suggests how the distant mutations might indirectly change the substrate-binding site . Since proteins contain many more residues far from the active site than close to the active site, random mutagenesis is strongly biased in favor of distant mutations . Directed evolution rarely screens all mutations, so it usually finds the distant mutations because they are more common, but probably not the most effective.

J Aerosol Med, 2003 Spring, 16(1), 55 - 64
Evaluation of the survival of bacterial contaminants in an inhalable insulin powder; Adhikari A et al.; Survival and growth of three model test bacterial species (Pseudomonas fluorescens, Staphylococcus epidermidis and Bacillus subtilis), present in the air and/or in the human respiratory tract, were tested in inhalable insulin-lactose powder under optimal relative humidity and temperature conditions (RH = 96% and optimal growth temperature for each bacterium of 26-37 degrees C) as well as representative indoor conditions (RH = 43% and T = 20 degrees C) . The bacteria survived from 12 h to 7 days depending on the bacterial species and the test condition . P . fluorescens vegetative cells had the lowest and B . subtilis spores the highest survival rate . It was found that insulin-lactose powder does not support bacterial growth and that higher bacterial survival rate was found under representative indoor conditions . Selected experiments were performed with B . subtilis by adding sterile saliva into insulin-lactose powder to represent a typical condition for inhaler use . Furthermore, two other powders were tested with B . subtilis: one representing an inert powder without any nutrients (glass beads) and the other representing a powder with optimal nutrients (tryptic soy broth powder) . The data indicate that the survival rate of B . subtilis did not change after the saliva was added and that the survival in insulin-lactose powder was similar to that in inert powder but lower than in powder with optimal nutrients . These results suggest that bacterial growth on residual powder in the inhaler under patient use conditions is unlikely and therefore the concern for patient safety is remote.

J Antibiot (Tokyo), 2003 Feb, 56(2), 68 - 71
FR252921, a novel immunosuppressive agent isolated from Pseudomonas fluorescens no . 408813 III . In vivo activities; Fujine K et al.; A novel immunosuppressive agent, FR252921 was isolated from the cultured broth of a species of Pseudomonas fluorescens . We have shown that FR252921 inhibit activating protein-1 (AP-1) transcription activity and act dominantly against antigen presenting cells comparing to T cell . Possibility of FR252921 as concomitant drug of FK506, T-cell specific inhibitor was evaluated . FR252921 showed synergy with FK506 in immunosuppressive activity both in splenic proliferation and in murine skin transplantation.

J Antibiot (Tokyo), 2003 Feb, 56(2), 62 - 7
FR252921, a novel immunosuppressive agent isolated from Pseudomonas fluorescens no . 408813 II . In vitro property and mode of action; Fujine K et al.; A novel immunosuppressive agent, FR252921 was isolated from the cultured broth of a species of Pseudomonas fluorescens . We have shown that FR252921 inhibited splenic proliferation stimulated with LPS, insensitive to calcinuerin inhibitor . In this study, FR252921 was found to inhibit IL-2 and IL-12 production as well as proliferaion of splenocyte . Analysis of transcription activity revealed that FR252921 inhibited activating protein-1 (AP-1) . Exposures of antigen presenting cells (APC) to FR252921 attenuated proliferation supplemented by naive T cells . Further, FR252921 strongly suppressed splenic dendritic cell proliferation stimulated with LPS and anti-CD40 mAb, while it did not inhibit purified T cell activation, including CD154 expression and IL-2 production . These results suggest that APC is dominant target cell population.

J Antibiot (Tokyo), 2003 Feb, 56(2), 55 - 61
FR252921, a novel immunosuppressive agent isolated from Pseudomonas fluorescens no . 408813 . I . Taxonomy, fermentation, isolation, physico-chemical properties and biological activities of FR252921, FR252922 and FR256523; Fujine K et al.; Novel immunosuppressive agents, FR252921, FR252922 and FR256523 were isolated from the cultured broth of a bacterial strain No . 408813 . The strain was identified Pseudomonas fluorescens from morphological and physiological characteristics . FR252921, FR252922 and FR256523, novel compounds containing macrolactone ring, showed immunosuppressive activity against murine splenocyte proliferation stimulated with lipopolysaccharide (LPS) or anti-CD3 mAb in vitro.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 393 - 400
Reclassification of 'Pseudomonas fluorescens subsp . cellulosa' NCIMB 10462 (Ueda et al . 1952) as Cellvibrio japonicus sp . nov . and revival of Cellvibrio vulgaris sp . nov., nom . rev . and Cellvibrio fulvus sp . nov., nom . rev; Humphry DR et al.; 'Pseudomonas fluorescens subsp . cellulosa' NCIMB 10462 has been demonstrated by a polyphasic taxonomic approach to be a member of the genus Cellvibrio . 16S rDNA sequence analysis suggests that this is the only genus that could accept this specimen . The sequence is 95.5% similar to that of Cellvibrio mixtus subsp . mixtus ACM 2601T (the type strain of the type species of the genus), which is its closest relation . The genomic DNA G + C content was determined to be 53.3 mol%, which is similar to the values obtained for the validly described Cellvibrio species . DNA-DNA hybridization experiments have shown that strain NCIMB 10462T (= NCDO 2697T) represents a novel species; therefore, it is proposed that it be designated as the type strain of the novel species Cellvibrio japonicus sp . nov . This study also used 16S rDNA analysis, DNA-DNA hybridization experiments and phenotypic testing to revive the species Cellvibrio vulgaris sp . nov., nom . rev . and Cellvibrio fulvus sp . nov., nom . rev . C . vulgaris NCIMB 8633T (=LMG 2848T) and C . fulvus NCIMB 8634T (=LMG 2847T) are the proposed type strains.

Res Microbiol, 2003 Apr, 154(3), 175 - 81
Equilibrium and kinetic adsorption of bacteria on alluvial sand and surface thermodynamic interpretation; Chen G et al.; Equilibrium and kinetic adsorption of Escherichia coli HB 101, E . coli JM 109, Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas sp . on alluvial sand from the Canadian River alluvium (Norman, OK) was investigated through column experiments . Equilibrium adsorption of these five bacterial strains followed the Freundlich expression and was a function of zero energy points, an indication of the zero energy buffer zone . Among the microorganisms studied, P . putida had the greatest equilibrium adsorption (162.4 x 10(8) cell/g sediment with a microbial injectate concentration of 10(8) cell/ml), followed by Pseudomonas sp . (127.9 x 10(8) cell/g sediment), E . coli HB 101 (62.8 x 10(8) cell/g sediment), E . coli JM 109 (58.4 x 10(8) cell/g sediment), and P . fluorescens (42.6 x 10(8) cell/g sediment) . The first-order kinetic adsorption rate coefficient was an exponential function of the total interaction free energy between the bacteria and sediment evaluated at the primary minimum, Delta G(132)(TOT) (PM) . E . coli HB 101 had the greatest kinetic adsorption rate coefficient on the sediment (5.10 h(-1)), followed by E . coli JM 109 (4.52 h(-1)), P . fluorescens (2.12 h(-1)), P . putida (2.04 h(-1)), and Pseudomonas sp . (1.34 h(-1)).

Appl Microbiol Biotechnol, 2003 May, 61(3), 240 - 6 Epub 2003 Jan 09.
Characterization of pNI10 plasmid in Pseudomonas, and the construction of an improved Escherichia and Pseudomonas shuttle vector, pNUK73; Itoh N et al.; The complete nucleotide sequence of pNI10 (3.75 kb), from which pNI105 and pNI107 were constructed as medium-host-range vectors for Gram-negative bacteria, was determined . A fragment of about 2.1 kb of pNI10 was essential for replication in Escherichia coli and Pseudomonas fluorescens . This fragment encodes a putative origin of replication ( ori) and one putative replication-controlling protein (Rep) . An improved version of the medium-host-range plasmid vector pNUK73 (5.13 kb) was constructed with the basic-replicon of pNI10 and pHSG298 (2.68 kb) . We show that expression in pseudomonads of the bromoperoxidase gene ( bpo) of Pseudomonas putida, inserted downstream of the lac promoter in pNUK73, resulted in about 30% (13.6 U/l culture) of the enzyme level obtained in E . coli.

Indian J Exp Biol, 2002 Oct, 40(10), 1131 - 6
Biodegradation of benzidine based azodyes Direct red and Direct blue by the immobilized cells of Pseudomonas fluorescens D41; Puvaneswari N et al.; Benzidine based azodyes are proven carcinogens, mutagens and have been linked to bladder cancer of human beings and laboratory animals . The textile and dyestuff manufacturing industry are the two major sources that released azodyes in their effluents . The dye, Direct blue contains two carcinogenic compounds namely benzidine (BZ), 4-amino biphenyl (4-ABP), while the dye Direct red has benzidine (BZ) . Among 40 isolates of Pseudomonas fluorescens screened, one isolate designated as D41 was found to be capable of extensively degrading the dyes Direct blue and Direct red . Immobilized cells of P . fluorescens D41 efficiently degraded Direct red (82%) and Direct blue (71%) in the presence of glucose.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 310 - 4
The D-glucosaminate dehydratase alpha-subunit from Pseudomonas fluorescens exhibits thioredoxin reductase activity; Iwamoto R et al.; The complete amino acid sequence of the D-glucosaminate dehydratase (GADH) alpha-subunit from Pseudomonas fluorescens was determined by PCR using genomic DNA from P . fluorescens as a template . The alpha-subunit comprises 320 amino acids and has a molecular mass of about 33.9 kDa . The primary structure of the alpha-subunit demonstrates a high similarity to the structures of thioredoxin reductase (TrxR) from many prokaryotes, especially Pseudomonas aeruginosa (identity 85%, positive 91%), Vibrio cholerae (identity 73%, positive 85%), and Escherichia coli (identity 71%, positive 83%) . The purified glucosaminate dehydratase alpha(2)-enzyme exhibited NADPH-dependent TrxR activity, while TrxR from E . coli showed pyridoxal 5'-phosphate (PLP)-dependent GADH activity . The TrxR from E . coli suggests that there are three cofactor binding sites, FAD, NADPH, and PLP in the enzyme and that TrxR catalyzes the FAD- and NADPH-dependent oxidation-reduction reaction and the PLP-dependent alpha,beta-elimination reaction.

Appl Environ Microbiol, 2003 Apr, 69(4), 2023 - 31
Isolation and antifungal and antioomycete activities of aerugine produced by Pseudomonas fluorescens strain MM-B16; Lee JY et al.; The bacterial strain MM-B16, which showed strong antifungal and antioomycete activity against some plant pathogens, was isolated from a mountain forest soil in Korea . Based on the physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, the bacterial strain MM-B16 was identical to Pseudomonas fluorescens . An antibiotic active against Colletotrichum orbiculare and Phytophthora capsici in vitro and in vivo was isolated from the culture filtrates of P . fluorescens strain MM-B16 using various chromatographic procedures . The molecular formula of the antibiotic was deduced to be C(10)H(11)NO(2)S (M(+), m/z 209.0513) by analysis of electron impact mass spectral data . Based on the nuclear magnetic resonance and infrared spectral data, the antibiotic was confirmed to have the structure of a thiazoline derivative, aerugine {4-hydroxymethyl-2-(2-hydroxyphenyl)-2-thiazoline} . C . orbiculare, P . capsici, and Pythium ultimum were most sensitive to aerugine (MICs for these organisms were approximately 10 micro g ml(-1)) . However, no antimicrobial activity was found against yeasts and bacteria even at concentrations of more than 100 micro g ml(-1) . Treatment with aerugine exhibited a significantly high protective activity against development of phytophthora disease on pepper and anthracnose on cucumber . However, the control efficacy of aerugine against the diseases was in general somewhat less than that of the commercial fungicides metalaxyl and chlorothalonil . This is the first study to isolate aerugine from P . fluorescens and demonstrate its in vitro and in vivo antifungal and antioomycete activities against C . orbiculare and P . capsici.

J Biol Chem, 2003 Jul 11, 278(28), 25887 - 94 Epub 2003 Apr 02.
How does Pseudomonas fluorescens avoid suicide from its antibiotic pseudomonic acid?: Evidence for two evolutionarily distinct isoleucyl-tRNA synthetases conferring self-defense; Yanagisawa T et al.; Two isoleucyl-tRNA synthetases (IleRSs) encoded by two distinct genes (ileS1 and ileS2) were identified in pseudomonic acid (mupirocin)-producing Pseudomonas fluorescens . The most striking difference between the two IleRSs (IleRS-R1 and IleRS-R2) is the difference in their abilities to resist pseudomonic acid . Purified IleRS-R2 showed no sensitivity to pseudomonic acid even at a concentration of 5 mm, 105 times higher than the Ki value of IleRS-R1 . The amino acid sequence of IleRS-R2 exhibits eukaryotic features that are originally found in eukaryotic proteins . Escherichia coli cells transformed with the ileS2 gene exerted pseudomonic acid resistance more than did those transformed with ileS1 . Cells transformed with both genes became almost as resistant as P . fluorescens . These results suggest that the presence of IleRS-R2 could be the major reason why P . fluorescens is intrinsically resistant to the antibiotic . Here we suggest that the evolutionary scenario of the eukaryotic ileS2 gene can be explained by gene acquisition and that the pseudomonic acid producer may have maintained the ileS2 gene to protect itself from pseudomonic acid.

Biochem Biophys Res Commun, 2003 Apr 11, 303(3), 926 - 31
Styrene-catabolism regulation in Pseudomonas fluorescens ST: phosphorylation of StyR induces dimerization and cooperative DNA-binding; Leoni L et al.; Styrene is an important chemical extensively used in the petrochemical and polymer industries . In Pseudomonas fluorescens ST, styrene metabolism is controlled by a two-component regulatory system, very uncommon in the degradation of aromatic compounds . The two-component regulatory proteins StyS and StyR regulate the expression of the styABCD operon, which codes for styrene degradation . StyS corresponds to the sensor kinase and StyR to the response regulator, which is essential for the activation of PstyA, the promoter of the catabolic operon . In two-component systems, the response regulator is phosphorylated by the cognate sensor kinase . Phosphorylation activates the response regulator, inducing DNA-binding . The mechanism underlying this activation has been reported only for a very few response regulators . Here, the effect of phosphorylation on the oligomeric state and on the DNA-binding properties of StyR has been investigated . Phosphorylation induces dimerization of StyR, the affinity of dimeric StyR for the target DNA is higher than that of the monomer, moreover dimeric StyR binding to the DNA target is cooperative . Furthermore, StyR oligomerization may be driven by the DNA target . This is the first direct demonstration that StyR response regulator binds to the PstyA promoter.

Chemosphere, 2003 Jun, 51(8), 701 - 6
A sulfonic anhydride derivative from dibenzyl trisulphide with agro-chemical activities; Williams LA et al.; In the present study, the biologically active natural product dibenzyl trisulphide (DTS) which was previously isolated from the sub-tropical shrub Petiveria alliacea was transformed to methyl benzyl sulphonic anhydride (MBSA) using a "one pot" transformation method . The anhydride was evaluated for anti-microbial activities on the bacteria, Bacillus subtilis and Pseudomonas fluorescens and found to be 2.5 fold more effective than the commercial agents isoniazid and ampicillin in inhibiting the growth of B . subtilis, while on P . fluorescens it was 2.5, 5.0 and 10.0 fold more inhibitory than isoniazid, ampicillin and dibenzyl trisulphide, respectively . DTS was inactive on B . subtillis . The MIC value (microgram/spot) found for DTS on the plant pathogenic fungus, Cladosporium cucumerinum was 5.0 microgram/spot, while MBSA gave a value of 0.1 microgram/spot, compared with 1.25 and 0.16 microgram/spot for the commercial agents ketoconazole and nystatin, respectively . On the larval nematode (Meloidogyne incognita) MBSA inflicted 97.72% and 57.47% Abbotts nematicidal activities at 125.0 and 62.5 ppm, respectively, while DTS had no effect at 125.0 ppm . Nematodes which were immobilized by the low concentrations of MBSA were unable to re-activate when exposed to 10.0 ppm picrotoxin, thus suggesting that the anhydride nematicidal activity is independent of the GABA-ergic neurophysiological pathway.MBSA demonstrated a strong dose dependent radicular suppression effect (r=0.984), on the radicles of Latuca sativa germinating seeds . DTS was weakly active.

Regul Toxicol Pharmacol, 2003 Feb, 37(1), 149 - 68
Safety evaluation of an alpha-amylase enzyme preparation derived from the archaeal order Thermococcales as expressed in Pseudomonas fluorescens biovar I; Landry TD et al.; BD5088 alpha-amylase derived from archaeal sources has characteristics of pH and temperature tolerance that are well suited to hydrolysis of starch in food processing applications . The production microorganism recipient strain, Pseudomonas fluorescens biovar I, strain MB101, was avirulent after oral administration to mice and does not represent an infectious threat to humans . Repeated dose gavage studies with BD5088 enzyme preparation, up to 13 weeks in duration, showed no systemic toxicity due to the oral route with an NOAEL of 890 mg/kg/day as Total Organic Solids . Some irritation occurred in the respiratory tract, which was considered to be a consequence of reflux and aspiration of test material that contained lipopolysaccharide from the Pseudomonas production strain . A 2-week dietary study (0 and 310 mg/kg/day) confirmed that there were no respiratory tract effects related to oral ingestion . There was no genotoxic activity based on Ames, mouse lymphoma, mouse micronucleus, and rat lymphocyte chromosomal aberration tests . There was no evidence of allergenic potential based on a comparison of the primary sequence of BD5088 with sequences in an allergen database . The enzyme was labile to pepsin digestion . Based on these data, BD5088 alpha-amylase preparation may be considered safe for use in food production such as corn wet milling .

Biochim Biophys Acta, 2003 Apr 1, 1611(1-2), 223 - 33
Protein secretion systems of Pseudomonas aeruginosa and P fluorescens; Ma Q et al.; Gram-negative bacteria have evolved numerous systems for the export of proteins across their dual-membrane envelopes . Three of these systems (types I, III and IV) secrete proteins across both membranes in a single energy-coupled step . Four systems (Sec, Tat, MscL and Holins) secrete only across the inner membrane, and four systems {the main terminal branch (MTB), fimbrial usher porin (FUP), autotransporter (AT) and two-partner secretion families (TPS)} secrete only across the outer membrane . We have examined the genome sequences of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens Pf0-1 for these systems . All systems except type IV were found in P . aeruginosa, and all except types III and IV were found in P . fluorescens . The numbers of each such system were variable depending on the system and species examined . Biochemical and physiological functions were assigned to these systems when possible, and the structural constituents were analyzed . Available information regarding the mechanisms of transport and energy coupling as well as physiological functions is summarized . This report serves to identify and characterize protein secretion systems in two divergent pseudomonads, one an opportunistic human pathogen, the other a plant symbiont.

Biochem Biophys Res Commun, 2003 Apr 4, 303(2), 631 - 9
Crystal structure of a histidine-tagged serine hydrolase involved in the carbazole degradation (CarC enzyme); Habe H et al.; 2-Hydroxy-6-oxo-6-(2(')-aminophenyl)-hexa-2,4-dienoate hydrolases (CarC enzymes) from two carbazole-degrading bacteria were purified using recombinant Escherichia coli strains with the histidine (His)-tagged purification system . The His-tagged CarC (ht-CarC) enzymes from Pseudomonas resinovorans strain CA10 (ht-CarC(CA10)) and Janthinobacterium sp . strain J3 (ht-CarC(J3)) exhibited hydrolase activity toward 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate as the purified native CarC(CA10) did . ht-CarC(J3) was crystallized in the space group I422 with cell dimensions of a=b=130.3A, c=84.5A in the hexagonal setting, and the crystal structure of ht-CarC(J3) was determined at 1.86A resolution . The final refined model of ht-CarC(J3) yields an R-factor of 21.6%, although the electron-density corresponding to Ile146 to Asn155 was ambiguous in the final model . We compared the known structures of BphD from Rhodococcus sp . strain RHA1 and CumD from Pseudomonas fluorescens strain IP01 . The backbone conformation of ht-CarC(J3) was better superimposed with CumD than with BphD(RHA1) . The side-chain directions of Arg185 and Trp262 residues in the substrate binding pockets of these enzymes were different among these proteins, suggesting that these residues may take a conformational change during the catalytic cycles.

Chemosphere, 2003 Jan, 50(3), 409 - 13
Toxicity and bioavailability to bacteria of particle-associated arsenite and mercury; Petanen T et al.; The overall toxicity of soil, and the bioavailability and arsenite from soil were measured with the constructed constitutively luminescent strain Pseudomonas fluorescens OS8 (pNEP01) and with earlier published biosensor strains P . fluorescens OS8 (pTPT11) for mercury and P . fluorescens OS8 (pTPT31) for arsenite, respectively . Both spiked and authentic samples were studied . By combining bacterial assays enabled partial analysis of reasons for toxicity of environmental samples, some of which were highly toxic despite containing little or no heavy metals . The spiked soils were not toxic overall but the method of measuring concentration from water-extractable fraction or from soil-water slurry affected the results significantly . Mercury that was bound to clay even after water extraction was nevertheless found to be bioavailable to a high degree to the biosensor bacteria . Since induction of the luminescence genes takes place intracellularly the bacteria may able to apparently release mercury when in direct contact with clay particle . This type of biomobilisation was not observed with arsenite spiked soils . The same phenomenon was detected in one of the environmental samples.

DNA Seq, 2002 Dec, 13(6), 343 - 51
Analysis of rILERS, an isoleucyl-tRNA synthetase gene associated with mupirocin production by Pseudomonas fluorescens NCIMB 10586; Rangaswamy V et al.; Some strains of Pseudomonas fluorescens produce the antibiotic mupirocin, which functions as a competitive inhibitor of isoleucyl-tRNA synthetase (ILERS) . Mupirocin-producing strains of P . fluorescens must overcome the inhibitory effects of the antibiotic to avoid self-suicide . However, it is not clear how P . fluorescens protects itself from the toxic effects of mupirocin . In this report, we describe a second gene encoding isoleucyl-tRNA synthetase (rILERS) in P . fluorescens that is associated with the mupirocin biosynthetic gene cluster . Random mutagenesis of the mupirocin-producing strain, P . fluorescens 10586, resulted in a mupirocin-defective mutant disrupted in a region with similarity to ILERS, the target site for mupirocin . The ILERS gene described in the present study was sequenced and shown to be encoded by a 3093 bp ORF, which is 264 bp larger than the ILERS gene previously identified in P . fluorescens 10586 . rILERS from P . fluorescens is most closely related to prokaryotic or eukaryotic sources of ILERS that are resistant to mupirocin . Interestingly, the relatedness between rILERS and the ILERS previously described in P . fluorescens 10586 was low (24% similarity), which indicates that P . fluorescens contains two isoforms of isoleucyl-tRNA synthetase.

Tree Physiol, 1998 Feb, 18(2), 103 - 111
In vitro effects of Laccaria bicolor S238 N and Pseudomonas fluorescens strain BBc6 on rooting of de-rooted shoot hypocotyls of Norway spruce; Karabaghli C et al.; The ectomycorrhizal fungus Laccaria bicolor S238 N and the bacterium Pseudomonas fluorescens BBc6 were used separately and in combination to induce in vitro rooting of de-rooted shoot hypocotyls of Norway spruce (Picea abies (L.) Karst.) . When the culture medium was supplemented with tryptophan, a precursor of indole-3-acetic acid (IAA) synthesis, the presence of the ectomycorrhizal fungus increased the percentage of hypocotyls forming roots; furthermore, both the fungal and bacterial inoculations enhanced the number of roots formed per rooted hypocotyl . Similar results were obtained by adding exogenous IAA (5 and 10 &mgr;M) to the rooting medium . After the rooting phase, the fungal inoculation enhanced adventitious root elongation and branching as well as the aerial growth of the cuttings . Pseudomonas fluorescens BBc6 had no effect on these parameters . The production of IAA by pure cultures of L . bicolor S238 N and P . fluorescens BBc6 was estimated by immunochemical analysis using specific anti-IAA antibodies . Both L . bicolor S238 N and P . fluorescens BBc6 synthesized IAA in pure culture and synthesis was stimulated in the presence of tryptophan . Thus, the effect of the fungus in stimulating adventitious root formation and subsequent elongation and branching can be attributed, at least partially, to the synthesis of IAA by the fungus . The finding that P . fluorescens BBc6 had no effect on root elongation and branching although it produced IAA suggests that either IAA was not the only parameter involved in the stimulation of these processes by L . bicolor S238 N or the bacterium produced other compounds that counteracted the stimulatory effects of IAA on root elongation and branching.

Protein Sci, 2003 Apr, 12(4), 681 - 9
Amphiphilic biopolymers (amphibiopols) as new surfactants for membrane protein solubilization; Duval-Terrie C et al.; The aim of this study was to develop new surfactants for membrane protein solubilization, from a natural, biodegradable polymer: the polysaccharide pullulan . A set of amphiphilic pullulans (HMCMPs), differing in hydrophobic modification ratio, charge ratio, and the nature of the hydrophobic chains introduced, were synthesized and tested in solubilization experiments with outer membranes of Pseudomonas fluorescens . The membrane proteins were precipitated, and then resolubilized with various HMCMPs . The decyl alkyl chain (C(10)) was the hydrophobic graft that gave the highest level of solubilization . Decyl alkyl chain-bearing HMCMPs were also able to extract integral membrane proteins from their lipid environment . The best results were obtained with an amphiphilic pullulan bearing 18% decyl groups (18C(10)) . Circular dichroism spectroscopy and membrane reconstitution experiments were used to test the structural and functional integrity of 18C(10)-solubilized proteins (OmpF from Escherichia coli and bacteriorhodopsin from Halobacterium halobium) . Whatever their structure type (alpha or beta), 18C(10) did not alter either the structure or the function of the proteins analyzed . Thus, HMCMPs appear to constitute a promising new class of polymeric surfactants for membrane protein studies.

FEMS Microbiol Lett, 2003 Mar 14, 220(1), 35 - 9
A tripartite microbial reporter gene system for real-time assays of soil nutrient status; Standing D et al.; Plant-derived carbon is the substrate which drives the rate of microbial assimilation and turnover of nutrients, in particular N and P, within the rhizosphere . To develop a better understanding of rhizosphere dynamics, a tripartite reporter gene system has been developed . We used three lux-marked Pseudomonas fluorescens strains to report on soil (1) assimilable carbon, (2) N-status, and (3) P-status . In vivo studies using soil water, spiked with C, N and P to simulate rhizosphere conditions, showed that the tripartite reporter system can provide real-time assessment of carbon and nutrient status . Good quantitative agreement for bioluminescence output between reference material and soil water samples was found for the C and P reporters . With regard to soil nitrate, the minimum bioavailable concentration was found to be greater than that analytically detectable in soil water . This is the first time that bioavailable soil C, N and P have been quantified using a tripartite reporter gene system.

Lett Appl Microbiol, 2003, 36(4), 239 - 44
Improvement of Pseudomonas fluorescens CHA0 biocontrol activity against root-knot nematode by the addition of ammonium molybdate; Hamid M et al.; AIMS: To improve the efficacy of Pseudomonas fluorescens CHA0 and its genetically modified (GM) derivatives by adding ammonium molybdate to control Meloidogyne javanica, the root-knot nematode in mungbean . METHODS AND RESULTS: Culture filtrate of P . fluorescens CHA0 and its GM derivative (antibiotic overproducing strain CHA0/pME3424 and antibiotic-deficient CHA89) obtained from nutrient broth yeast extract medium amended with 1, 2 or 4 mm of ammonium molybdate (NH4-Mo) caused substantial mortality of M . javanica juveniles in vitro . Pseudomonas fluorescens CHA0 or CHA0/pME3424 applied in conjunction with NH4-Mo caused greater reduction of nematode penetration in mungbean roots compared with the bacterial application alone . Ammonium molybdate at 4 mg kg-1 of soil along with CHA0 also enhanced plant height while shoot weight remained unaffected . Either used alone or in conjunction with NH4-Mo, strain CHA89 did not reduce nematode invasion compared with the controls . Bacterial strains did not differ significantly in their colonization potential in the mungbean rhizosphere . Efficacy of the biocontrol bacteria to control root-knot nematode was accentuated when soil was treated with NH4-Mo and zinc (both at 1 mg kg-1 of soil) . CONCLUSION: The addition of ammonium molybdate enhances the production of nematicidal compounds by P . fluorescensin vitro and improves bacterial efficacy against root-knot nematode under glasshouse conditions . SIGNIFICANCE AND IMPACT OF THE STUDY: Application of minerals such as ammonium molybdate is appealing because they are cheap and can easily be applied under field conditions to improve biocontrol potential of the bacterial inoculants . They also significantly reduce the amount of biocontrol inoculant biomass required to achieve root-knot disease control, with a consequent reduction in cost.

Mol Microbiol, 2003 Mar, 47(6), 1695 - 708
Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus; Aldridge P et al.; Several members of the two-component signal transduction family have been implicated in the control of polar development in Caulobacter crescentus: PleC and DivJ, two polarly localized histidine sensor kinases; and the response regulators DivK and PleD . The PleD protein was shown previously to be required during the swarmer-to-stalked cell transition for flagellar ejection and efficient stalk biogenesis . Here, we present data indicating that PleD also controls the onset of motility and a cell density switch immediately preceding cell division . Constitutively active alleles of pleD or wspR, an orthologue from Pseudomonas fluorescens, almost completely suppressed C . crescentus motility and inhibited the increase in swarmer cell density during cell differentiation . The observation that these alleles also had a dominant-negative effect on motility in a pleC divJ and a pleC divK mutant background indicated that PleD is located downstream of the other components in the signal transduction cascade, which controls the activity of the flagellar motor . In addition, the presence of a constitutive pleD or wspR allele resulted in a doubling of the average stalk length . Together, this is consistent with a model in which the active form of PleD, PleD approximately P, negatively controls aspects of differentiation in the late predivisional cell, whereas it acts positively on polar development during the swarmer-to-stalked cell transition . In agreement with such a model, we found that DivJ, which localizes to the stalked pole during cell differentiation, positively controlled the in vivo phosphorylation status of PleD, and the swarmer pole-specific PleC kinase modulated this status in a negative manner . Furthermore, domain switch experiments demonstrated that the WspR GGDEF output domain from P . fluorescens is active in C . crescentus, favouring a more general function for this novel signalling domain over a specific role such as DNA or protein interaction . Possible roles for PleD and its C-terminal output domain in modulating the polar cell surface of C . crescentus are discussed.

Z Naturforsch {C}, 2003 Jan-Feb, 58(1-2), 1 - 10
The pyoverdin of Pseudomonas fluorescens G173, a novel structural type accompanied by unexpected natural derivatives of the corresponding ferribactin; Fernandez DU et al.; The siderophores produced by Pseudomonas fluorescens G173 are unusual in several respects . So far all pyoverdins with a C-terminal cyclopeptidic substructure have in common that the epsilon-amino group of an in-chain Lys is bound amidically to the carboxyl group of a C-terminal Ser or Thr and that N5-formyl-N5-hydroxy Orn (FoOHOrn) is the next amino acid after Lys . FoOHOrn may (cyclotetrapeptidic structures) be or may not (cyclotripeptidic structures) be followed by a further amino acid . In the pyoverdin described here Orn instead of Lys is the amino acid forming the cycle, FoOHOrn is replaced by AcOHOrn which does not follow the branching Orn but is the penultimate amino acid and finally the last amino acid is Asp . The producing strain which had been classified as Pseudomonas fluorescens may well be a new species . Pyoverdins are frequently accompanied by ferribactins which are considered to be their biogenetic precursors . They always have the same amino acid chain as the co-occurring pyoverdins but the pyoverdin chromophore is replaced by a condensation product of L-Dab and D-Tyr with the amino group of Tyr bound to the gamma-carboxyl group of Glu . A ferribactin having these structural characteristics is produced by the investigated strain, but it is accompanied by derivatives where the alpha-amino group of Glu is partially or completely transformed into a hydroxamic acid by substitution with a hydroxyl and/or acetyl group.

Appl Environ Microbiol, 2003 Mar, 69(3), 1827 - 31
The competitiveness of Pseudomonas chlororaphis carrying pJP4 is reduced in the Arabidopsis thaliana rhizosphere; Schmidt-Eisenlohr H et al.; The effect of the large catabolic IncP plasmid pJP4 on the competitiveness of Pseudomonas chlororaphis SPR044 and on its derivatives SPR244 (GacS deficient), SPR344 (phenazine-1-carboxamide overproducer), and SPR644 (phenazine-1-carboxamide deficient) in the Arabidopsis thaliana rhizosphere was assessed . Solitary rhizosphere colonization by the wild type, SPR244, and SPR644 was not affected by the plasmid . The size of the population of SPR344 carrying pJP4, however, was significantly reduced compared to the size of the population of the plasmid-free derivative . The abiotic stress caused by phenazine-1-carboxamide overproduction probably resulted in a selective disadvantage for cells carrying pJP4 . Next, the effect of biotic stress caused by coinoculation of other bacteria was analyzed . Cells carrying pJP4 had a selective disadvantage compared to plasmid-free cells in the presence of the efficient colonizer Pseudomonas fluorescens WCS417r . This effect was not observed after coinoculation with a variety of other bacteria, and it was independent of quorum sensing and phenazine-1-carboxamide production . Thus, the presence of large catabolic plasmids imposes a detectable metabolic burden in the presence of biotic stress . Plasmid transfer in the A . thaliana rhizosphere from P . chlororaphis and its derivatives to Ralstonia eutropha was determined by using culture-dependent and culture-independent techniques . With the cultivation-independent technique we detected a significantly higher portion of exconjugants, but pJP4 transfer was independent of the quorum-sensing system and of phenazine-1-carboxamide production.

Appl Environ Microbiol, 2003 Mar, 69(3), 1564 - 72
Prokaryotic homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase in the 2-nitrobenzoate degradation pathway of Pseudomonas fluorescens strain KU-7; Muraki T et al.; The 2-nitrobenzoic acid degradation pathway of Pseudomonas fluorescens strain KU-7 proceeds via a novel 3-hydroxyanthranilate intermediate . In this study, we cloned and sequenced a 19-kb DNA locus of strain KU-7 that encompasses the 3-hydroxyanthranilate meta-cleavage pathway genes . The gene cluster, designated nbaEXHJIGFCDR, is organized tightly and in the same direction . The nbaC and nbaD gene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, respectively . The NbaC enzyme carries out the oxidation of 3-hydroxyanthranilate to 2-amino-3-carboxymuconate-6-semialdehyde, while the NbaD enzyme catalyzes the decarboxylation of the latter compound to 2-aminomuconate-6-semialdehyde . The NbaC and NbaD proteins were overexpressed in Escherichia coli and characterized . The substrate specificity of the 23.8-kDa NbaC protein was found to be restricted to 3-hydroxyanthranilate . In E . coli, this enzyme oxidizes 3-hydroxyanthranilate with a specific activity of 8 U/mg of protein . Site-directed mutagenesis experiments revealed the essential role of two conserved histidine residues (His52 and His96) in the NbaC sequence . The NbaC activity is also dependent on the presence of Fe(2+) but is inhibited by other metal ions, such as Zn(2+), Cu(2+), and Cd(2+) . The NbaD protein was overproduced as a 38.7-kDa protein, and its specific activity towards 2-amino-3-carboxymuconate-6-semialdehyde was 195 U/mg of protein . Further processing of 2-aminomuconate-6-semialdehyde to pyruvic acid and acetyl coenzyme A was predicted to proceed via the activities of NbaE, NbaF, NbaG, NbaH, NbaI, and NbaJ . The predicted amino acid sequences of these proteins are highly homologous to those of the corresponding proteins involved in the metabolism of 2-aminophenol (e.g., AmnCDEFGH in Pseudomonas sp . strain AP-3) . The NbaR-encoding gene is predicted to have a regulatory function of the LysR family type . The function of the product of the small open reading frame, NbaX, like the homologous sequences in the nitrobenzene or 2-aminophenol metabolic pathway, remains elusive.

Can J Microbiol, 2002 Dec, 48(12), 1069 - 75
Enhancement of population densities of fluorescent pseudomonads in the rhizosphere of tomato plants by addition of acibenzolar-S-methyl; Fakhouri WD et al.; Fluorescent pseudomonad isolates G309 and CW2, in combination with the resistance inducer acibenzolar-S-methyl (ASM), improved control of fungal and bacterial diseases on tomato plants . The interactions of the bacteria in the presence of ASM showed that in vitro growth of Pseudomonas fluorescens G309 and Pseudomonas sp . strain CW2 was not affected in King's B broth supplemented with 10 and 20 microM ASM . Also, the bacterial cells were not able to utilize ASM as a nutrient source . In vitro production of the two antimicrobial secondary metabolites phenazine-1-carboxylic acid and 2-OH-phenazine by the isolate CW2 was not affected within 3 days from incubation . In contrary, addition of ASM at a concentration of 20 microM to King's B liquid medium significantly increased production of salicylic acid by isolate G309 . When roots of tomato plants were treated with G309 or CW2 cell suspensions containing 20 microM ASM, the number of bacterial cells recovered from the rhizosphere was significantly higher in the combined treatments than in the single applications 5, 10, and 15 days after inoculation . However, ASM at a higher concentration (50 microM) did not appreciably enhance the population sizes of either bacterial isolate in the rhizosphere . Enhanced bacterial cell densities in the rhizosphere of tomato plants were also determined following simultaneous treatments of tomato roots with 10 and 20 microM ASM in combination with the transformed isolate G309-384 (mini-Tn5gfp), which encodes the green fluorescent protein.

J Environ Sci Health B, 2003 Mar, 38(2), 121 - 32
Degradation of 3,4-dichloro- and 3,4-difluoroaniline by Pseudomonas fluorescens 26-K; Travkin VM et al.; 3,4-Dichloro- and 3,4-difluoroanilines were degraded by Pseudomonas fluorescens 26-K under aerobic conditions . In the presence of glucose strain degraded 170 mg/L of 3,4-dichloroaniline (3,4-DCA) during 2-3 days . Increasing of toxicant concentration up to 250 mg/L led to degradation of 3,4-DCA during 4 days and its intermediates during 5-7 days . Without cosubstrate and nitrogen source degradation of 3,4-DCA took place too, but more slowly--about 40% of toxicant at initial concentration 75 mg/L was degraded during 15 days . 3,4-Difluoroaniline (3,4-DFA) (initial concentration 170 mg/L) was degraded by Pseudomonas fluorescens 26-K during 5-7 days . The strain was able to completely degrade up to 90 mg/L of 3,4-DFA, without addition of cosubstrate and nitrogen during 15 days . Degradation of fluorinated aniline was accompanied by intensive defluorination . Activity of catechol 2,3-dioxygenase (C2,3DO) (0.230 micromol/min/mg of protein) was found in the culture liquid of the strain, grown with 3,4-DCA and glucose . This fact, as well as, the presence of 3-chloro-4-hydroxyaniline as a metabolite suggested that 3,4-DCA degradation pathway includes dehalogenation and hydroxylation of aromatic ring followed by its subsequent cleaving by C2,3DO . On the contrary, activity of catechol 1,2-dioxygenase (C1,2DO) (0.08 micromol/min/mg of protein) was found in the cell-free extract of biomass grown on 3,4-DFA . 3-Fluoro-4-hydroxyaniline as intermediate was found in this cell-free extract.

Chem Biol Interact, 2003 Feb 1, 143-144, 559 - 82
Pseudomonas fluorescens mannitol 2-dehydrogenase and the family of polyol-specific long-chain dehydrogenases/reductases: sequence-based classification and analysis of structure-function relationships; Klimacek M et al.; Multiple sequence alignment and analysis of evolutionary relationships have been used to characterize a family of polyol-specific long-chain dehydrogenases/reductases (PSLDRs) . At the present time, 66 known and putative NAD(P)H-dependent oxidoreductases of mainly prokaryotic origin and between 357 and 544 amino acids in length constitute this family . The family is shown to include D-mannitol 2-dehydrogenase, D-mannonate 5-oxidoreductase, D-altronate 5-oxidoreductase, D-arabinitol 4-dehydrogenase, and D-mannitol-1-phosphate 5-dehydrogenase which form individual sub-families (defined by internal sequence identity of >/=30%) having distant origin and divergent substrate specificity but clearly displaying entire-chain relationship . When all forms are aligned, only three residues, Gly-33, Asp-230, and Lys-295 (in the numbering of Pseudomonas fluorescens D-mannitol 2-dehydrogenase (PsM2DH)) are strictly conserved . By combining sequence alignment with the known structure of PsM2DH and results from site-directed mutagenesis, we have developed a structure/function analysis for the family . Gly-33 is in the N-terminal coenzyme-binding domain and part of a nucleotide fingerprint region for the family, and Asp-230 and Lys-295 are at an interdomain segment contributing to the active site in which the lysine likely functions as the catalytic general acid/base . PSLDRs do not require a metal cofactor for activity and are specific for transferring the 4-pro-S hydrogen from NAD(P)H . Comparisons reveal that the core part of the two-domain fold has been conserved throughout all family members, perhaps reflecting the recruitment of a stable oxidoreductase structure and extensive trimming thereof to acquire functional properties specific to each sub-family . They also identify interactions that define the chemical mechanism of oxidoreduction and likely contribute to substrate and co-substrate specificities and are thus relevant for protein engineering.

Chem Biol Interact, 2003 Feb 1, 143-144, 551 - 8
Crystal structure of Pseudomonas fluorescens mannitol 2-dehydrogenase: evidence for a very divergent long-chain dehydrogenase family; Kavanagh KL et al.; Mannitol 2-dehydrogenase from Pseudomonas fluorescens (pfMDH) is a secondary alcohol dehydrogenase that catalyzes the reversible NAD(P)-dependent oxidation of D-mannitol to D-fructose, D-arabinitol to D-xylulose, and D-sorbitol to L-sorbose . It is a member of the mostly prokaryotic family of long-chain mannitol dehydrogenases that so far includes 66 members . Unlike other alcohol and polyol dehydrogenases that utilize metal cofactors or a conserved active-site tyrosine for catalysis, an invariant lysine is the general base . The crystal structure of pfMDH in a binary complex with NAD(H) and a ternary complex with NAD(H) and D-mannitol have been determined to 1.7 and 1.8 A resolution respectively . Comparison of secondary structure assignment to sequence alignments suggest the shortest members of this family, mannitol-1-phosphate 5-dehydrogenases, retain core elements but lack secondary structural components found on the surface of pfMDH . The elements predicted to be absent are distributed throughout the primary sequence, implying that a simple truncation or fusion did not occur . The closest structural neighbors are 6-phosphogluconate dehydrogenase, UDP-glucose dehydrogenase, N-(1-D-carboxyethyl)-L-norvaline dehydrogenase, and glycerol-3-phosphate dehydrogenase . Although sequence identity is only a barely recognizable 7-10%, conservation of secondary structural elements as well as homologous residues that are contributed to the active site indicates they may be related by divergent evolution.

Rev Argent Microbiol, 2002 Oct-Dec, 34(4), 193 - 8
Fluorescent Pseudomonas species causing post-harvest decay of endives in Argentina; Alippi AM et al.; A post-harvest bacterial decay was observed on ready-to-use French endives in Argentina . Affected chicons showed browning and soft-rot of inner leaves and marginal necrosis . Physiological and biochemical tests allowed us to identify the isolates from endive as Pseudomonas fluorescens bv . III, Pseudomonas fluorescens bv . V, and Pseudomonas cichorii . Pathogenicity was verified on RTU healthy endives by inoculation with each bacterial species, and also with the mixture of the 3 strains . P . cichorii caused dark brown necrosis of the margins of outer leaves; both isolates of P . fluorescens caused browning and soft-rotting of inner leaves, while the mixture induced all the described symptoms, that were similar to those found in natural infection . Identity of bacterial isolates was confirmed by RFLP analysis of a PCR-DNA fragment amplified from the 16S rRNA gene . This is the first record of a post-harvest decay in endives in Argentina.

Phytochemistry, 2003 Apr, 62(7), 1105 - 14
Structure, chemistry, and biological activity of pseudophomins A and B, new cyclic lipodepsipeptides isolated from the biocontrol bacterium Pseudomonas fluorescens; Pedras MS et al.; Pseudophomins A and B are cyclic lipodepsipeptides isolated from Pseudomonas fluorescens strain BRG100, a bacterium with potential application for biocontrol of plant pathogens and weeds . Their chemical structures were established by a combination of spectroscopic data, X-ray crystallography, and selective chemical degradation . This unique chemical degradation allowed the unambiguous determination of the absolute configuration of the amino acid residue Leu-1, due to gamma-lactam formation followed by selective cleavage of the adjacent N(8)-C(7) bond . To the best of our knowledge this is the first application of gamma-lactam formation to the determination of absolute configuration of an adjacent amino acid . Pseudophomin B showed higher antifungal activity against the phytopathogens Phoma lingam/Leptosphaeria maculans and Sclerotinia sclerotiorum than pseudophomin A, and is likely to be the main component responsible for the antifungal activity of EtOAc extracts of strain BRG100 . By contrast, pseudophomin A showed stronger inhibition of green foxtail (Setaria viridis) root germination than pseudophomin B.

Biotechnol Bioeng, 2003 Apr 20, 82(2), 232 - 7
Self-assembly of Pseudomonas fluorescens lipase into bimolecular aggregates dramatically affects functional properties; Fernandez-Lorente G et al.; It has been found that lipase from Pseudomonas fluorescens (PFL) is able to aggregate into bimolecular structures (MW around 66 kD) even at moderate enzyme concentrations . At very low enzyme concentrations and in the presence of detergents, the same enzyme displayed a unimolecular structure with a molecular weight of 33 kD . Both enzyme structures displayed different functional properties . First, the bimolecular structure was much more stable than the unimolecular species (the bimolecular structure maintained over 80% of initial activity after 72 hours at 45 degrees C, while the unimolecular structure retained only around 30% of initial activity after 4 hours of incubation under the same experimental conditions); and the bimolecular form presented a higher optimal T . Second, the unimolecular form showed a much lower K(M) for ethyl butyrate than the bimolecular form . Third, the interfacial activation in biphasic substrate-aqueous milieu was higher for the bimolecular form . Fourth, the unimolecular structure was less active but much more enantioselective than the unimolecular species in the model reaction used . It is proposed that the bimolecular aggregates of PFL might be formed by two open lipase molecules (mutual interfacial activation), in intimate contact, and that the bimolecular form represents an example of "pseudo-quaternary" structure .

Biometals, 2003 Jun, 16(2), 263 - 70
The pyoverdine from Pseudomonas chlororaphis D-TR133 showing mutual acceptance with the pyoverdine of Pseudomonas fluorescens CHAO; Barelmann I et al.; From Pseudomonas chlororaphis D-TR 133 a pyoverdine was isolated and its primary structure were elucidated by spectroscopic methods and degradation reactions . Despite some structural differences, its Fe(III) complex and that of the pyoverdine from Pseudomonas fluorescens CHA0 were taken up by either strain with a high rate . This is explained by a structural similarity between the two pyoverdines which were shown to differ in their structures only by the replacement of Lys by Ala in the C-terminal part of the molecules . An unexpected feature is that the main pyoverdine of P . chlororaphis D-TR133 is accompanied by a minor one where specifically one Ala is replaced by Gly . So far amino acid variations in the peptide chain of pyoverdines produced by a given strain had not been observed amongst the producers of the about fifty pyoverdines reported in the literature.

Appl Environ Microbiol, 2003 Feb, 69(2), 861 - 8
Production of cyclic lipopeptides by Pseudomonas fluorescens strains in bulk soil and in the sugar beet rhizosphere; Nielsen TH et al.; The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere . Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca . 5 microg g(-1)) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil . When a whole-cell inoculum of P . fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week . By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil . In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for approximately 2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days . All three CLPs remained detectable for several days in the rhizosphere . Subsequent tests of five other CLP-producing P . fluorescens strains also demonstrated significant production in the young rhizosphere . The results thus provide evidence that production of different CLPs is a common trait among many P . fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.

Transfusion, 2003 Feb, 43(2), 135 - 42
Prevention of Yersinia enterocolitica, Pseudomonas fluorescens, and Pseudomonas putida outgrowth in deliberately inoculated blood by a novel pathogen-reduction process; Zavizion B et al.; BACKGROUND: Yersinia enterocolitica, Pseudomonas fluorescens, and P . putida are responsible for a significant amount of the bacterial sepsis cases attributed to RBC transfusions . INACTINE is a pathogen-reduction process for RBCs, which consists of incubation of RBCs with PEN110 (proprietary compound) followed by automated washing of the RBCs . INACTINE is an electrophilic agent, which inactivates a wide range of viruses and WBCs by disruption of nucleic acid replication . The present study investigated the effect of the PEN110 process on Y . enterocolitica, P . fluorescens, and P . putida . STUDY DESIGN AND METHODS: Identical units of reduced CPD/ADSOL additive solution (AS-1) or CP2D/Nutricel additive solution (AS-3) RBCs were inoculated with 10 to 100 CFU per mL of either Y . enterocolitica, P . fluorescens, or P . putida . The control units were put on storage immediately after the bacterial spike . The test units were subjected to the PEN110 process and then stored . Sham control units were processed the same way as test units without addition of PEN110 . Bacterial titer in all units was monitored during the 6-week storage period . RESULTS: No bacteria were detected in any of the RBC units (n = 9 for each microorganism) prepared using the PEN110 process throughout 6 weeks of storage . Substantial bacterial growth occurred in all control and in a majority of sham control units (11 out of 15 experiments) . The bacterial inactivation by the INACTINE process was found to be equally effective in CPD/AS-1 and CP2D/AS-3 RBC units . CONCLUSION: The INACTINE process effectively prevented the outgrowth of Y . enterocolitica, P . fluorescens, and P . putida deliberately inoculated into WBC-reduced CPD/AS-1 and CP2D/AS-3 RBCs . The results demonstrated the crucial bactericidal role of PEN110 in the INACTINE process.

Environ Microbiol, 2003 Feb, 5(2), 103 - 15
Persistence and cell culturability of biocontrol strain Pseudomonas fluorescens CHA0 under plough pan conditions in soil and influence of the anaerobic regulator gene anr; Mascher F et al.; Certain fluorescent pseudomonads can protect plants from soil-borne pathogens, and it is important to understand how these biocontrol agents survive in soil . The persistence of the biocontrol strain Pseudomonas fluorescens CHA0-Rif under plough pan conditions was assessed in non-sterile soil microcosms by counting total cells (immunofluorescence microscopy), intact cells (BacLight membrane permeability test), viable cells (Kogure's substrate-responsiveness test) and culturable cells (colony counts on selective plates) of the inoculant . Viable but non-culturable cells of CHA0-Rif (106 cells g-1 soil) were found in flooded microcosms amended with fermentable organic matter, in which the soil redox potential was low (plough pan conditions), in agreement with previous observations of plough pan samples from a field inoculated with CHA0-Rif . However, viable but non-culturable cells were not found in unamended flooded, amended unflooded or unamended unflooded (i.e . control) microcosms, suggesting that such cells resulted from exposure of CHA0-Rif to a combination of low redox potential and oxygen limitation in soil . CHA0-Rif is strictly aerobic . Its anaerobic regulator ANR is activated by low oxygen concentrations and it controls production of the biocontrol metabolite hydrogen cyanide under microaerophilic conditions . Under plough pan conditions, an anr-deficient mutant of CHA0-Rif and its complemented derivative displayed the same persistence pattern as CHA0-Rif, indicating that anr was not implicated in the formation of viable but non-culturable cells of this strain at the plough pan.

Pest Manag Sci, 2003 Jan, 59(1), 21 - 4
Isolation of a Pseudomonas fluorescens metabolite/exotoxin active against both larvae and pupae of vector mosquitoes; Prabakaran G et al.; A formulation was developed from the metabolite(s) of a novel Pseudomonas fluorescens Migula strain (VCRC B426) and tested against 4th-instar larvae and pupae of three species of vector mosquitoes, Anopheles stephensi Liston, Culex quinquefasciatus Say and Aedes aegypti (L) . The larvae and pupae of An . stephensi were the most susceptible to the formulation, followed by those of C . quinquefasciatus and Ae . aegypti, in that order, and the dosage requirement for pupal mortality was less than that required for larval mortality . The LC50 dosage requirements for larvae of these mosquito species were, respectively, 70.4, 511.5 and 757.3 microg protein ml(-1), whereas for pupae they were, respectively, 2.0, 9.4 and 19.2 microg protein ml(-1) . The lethal fraction was purified from the culture broth and its molecular mass, as determined by high performance liquid chromatography, was 44kDa . This is the first report of a microbial formulation acting upon mosquito pupae, a non-feeding stage . Its mode of action and efficacy to control mosquitoes under field conditions need to be studied further.

Wei Sheng Wu Xue Bao, 1998 Feb, 38(1), 1 - 5
{Expression characteristic of Bacillus thuringiensis cry1 gene in Pseudomonas fluorescens Pfx-18}; Liu Z et al.; The plasmids of lepidopteran-specific Bacillus thuringiensis strain from our laboratory were hybridized with RNA probe of cry 1Aa EcoR I-F fragment labelled using DIG . Cry1 gene was located in 39.3 MD plasmid . The plasmid was digested with Hind III and analysed by southern blot . It appeared both 7.1 kb and of 6.5 kb positive bands . The 7.1 kb fragment was ligated to broad-host-range vector pSUP106 and transformed into Pseudomonas fluorescens Pfx-18 . The cloned strain, LZP-1 was obtained . The plasmids of LZP-1 were analysed by PCR . The results showed that gene-type is cry1Ab . SDS-PAGE analysis demonstrated that LZP-1 could express 66 kD insecticidal crystal protein and some small molecular weight peptides . Bioassay showed that motality of 1000-fold diluted fermentation broth was 33% to 3rd instar plulella xyloslelly larvae.

Wei Sheng Wu Xue Bao, 2001 Feb, 41(1), 3 - 8
{Some biological characteristics of genetically engineered insecticidal Pseudomonas fluorescens}; Ding Z et al.; Plasmid stability, Antifungal activity, plant-colonizing ability, UV resistance and inseticidal activity in field were analysed for the engineered Pseudomonas fluorescens (Pf) strain IPP202 . The results indicated that the recombinant plasmid in IPP202 was very stable after successive diluting culturing and after continuous culturing, There was no significant change in the properties beneficial to plants, such as antifungal activity and plant-colonizing ability as compared with the original strain P303 . IPP202 was much more resistant to UV than Bt strain HD73 . The control effect in field against cotton boll worm in field was close to that of a locally used Bt-chemical mixture in normal applied concentration . All the data indicated that the engineered Pf strain was a one with prosperous future after further study.

Wei Sheng Wu Xue Bao, 2000 Dec, 40(6), 573 - 8
{Expression and synergism of two cry insecticidal protein genes in Pseudomonas fluorescens}; Ding Z et al.; Several engineered Pseudomonas fluorescens(Pf) strains were constructed mainly based on a Pseudomonas plasmid pJMS6 alpha-lac and two insecticidal crystal protein genes of Bacillus thuringiensis, cry1Ac and cry2Aa, and the host Pf strain, P303, which was with highly antifungal activity to some plant disease fungi and colonizing ability on a wide range of plants . The DNA introduction was confirmed by PCR-RFLP and Southern blot . The 132 kD insecticidal protein was detected in IPP101 and IPP202 by SDS-PAGE and rhombic insecticidal protein crystals of them were observed through electron microscope, also indicating that cry1Ac gene was highly expressed . The results of insecticidal bioassay indicated that IPP101 was more toxic than IPP201, and IPP202 was the most toxic among the 3 strains . LC50 to the neonates of cotton boll worm(Helicoverpa armigera) were 0.02604, 0.00812 and 0.00186 mL/g feed, consecutively . In IPP202, two gene products showed significant synergism, with the co-toxicity coefficient of 332.8.

Wei Sheng Wu Xue Bao, 2000 Jun, 40(3), 312 - 7
{Effects of soil factors on root colonization of wheat by luxAB genes-marked Pseudomonas fluorescens Xl6L2}; Wang P et al.; Colonization density of Pseudomonas fluorescens Xl6L2 marked with luxAB genes in wheat Rhizosphere in sterilized Rhizobox--Calcareous chao soil microcosms was greater than that in insterilized microcosms . The population of Pf.Xl6L2 in the Rhizosphere in the rhizobox--Yellow brown soil microcosms was larger than that in the Calcareous chao soil microcosms; As soil water content was about 50% field capacity (FC), Pf.Xl6L2 could move to the place of 8 cm of root from coated seeds in the former microcosm, but only 4 cm in the later microcosm . In the former microcosms, the number of Pf.Xl612 in the rhizosphere under 60% FC and 75% FC were greater than that under 50% FC; In the second microcosm, the dispersal distance of Pf.Xl6L2 along the root was affected significantly by the soil moisture, it could be detected as far as 8 cm of root from the coated seeds under 60% FC and 75% FC.

Wei Sheng Wu Xue Bao, 2000 Apr, 40(2), 150 - 4
{Boot colonization of wheat by lux-AB genes marked Pseudomonas fluorescens Xl6L2}; Wang P et al.; Colonization density of Pseudomonas fluorescens Xl6L2 marked with luxAB genes in wheat rhizosphere in asepsis rhizobox-Calcareous chao soil microcosms reached the maximum(4.60 log cfu.g-1 root) 3 days after seeds coated with Pf.Xl6L2 sown, then declined to a relatively stable lower level(2.45 log cfu.g-1 root) in 0-2 cm root segment . Dynamics of colonization of Pf.Xl6L2 in wheat rhizosphere under field conditions was the same as in microcosms, Pf.Xl6L2 could move to the place of 10 cm of root from seeds under field conditions, distance of horizontal movement of Pf.Xl6L2 in field soil was not over 40 cm during 125 days of plant growth.

Environ Pollut, 2003, 122(3), 407 - 15
Assessing sediment toxicity and arsenite concentration with bacterial and traditional methods; Petanen T et al.; Three sediment samples LP (pool where logs are stored), LF (brook through landfill area), KN (Kaskesniemi) which is in Lake Pyhaselka downstream from the mill, were taken from an old sawmill area and one from the unpolluted Lake Hoytiainen . The arsenite concentration was measured by GFAAS and two arsenite biosensing bacterial strains Pseudomonas fluorescens OS8 (pTPT31) and Escherichia coli MC1061 (pTOO31) . The toxicity of sediment and pore water samples was determined by using luminescent bacteria (Flash test) and, further, whole sediment toxicity was measured using 10 days growth test and 50 days emergency test with midges (Chironomus riparius) . With the flash test a lowered EC50 value was found only in sediment LF (EC50=0.17 v/v%) . The Flash test indicated that all sediment samples taken from the sawmill area were highly toxic to bacteria, whereas growth and the emergence of chironomids showed no effects in other samples than LF . The midges tolerate well the contaminated environment . In contrast, bioavailability of arsenite of sediment samples KN and LF was quite high determined using the biosensor-strains in a direct contact assay . The bioavailable fraction of sediment LP was 6-10% out of the total arsenite concentration obtained with GFAAS (0.46-0.77 microg g-1 dw) . The results show that the choice of analysis method grossly affects the outcome without any of the method giving an incorrect result . Different methods measure different parameters of a toxic sample and can thus be used to complement each other.

Microb Ecol, 2003 Feb, 45(2), 145 - 55 Epub 2003 Jan 28.
Residual impact of the biocontrol inoculant Pseudomonas fluorescens F113 on the resident population of rhizobia nodulating a red clover rotation crop; Walsh UF et al.; A field trial was previously conducted in which sugarbeet seeds were either untreated, inoculated with the biocontrol strain Pseudomonas fluorescens F113Rif, or treated with chemical fungicides . Following harvest of sugarbeet, the field site was sown with uninoculated red clover . The aim of this study was to assess the residual impact of the microbial inoculant (and the fungicide treatment) on the diversity of resident rhizobia nodulating the red clover rotation crop . The percentage of nodules yielding rhizobial isolates after surface disinfection was 67% in the control and 70% in the P . fluorescens F113Rif treatment, but only 23% in the chemical treatment . Isolates were characterized by RAPD analysis . The main RAPD cluster (arbitrarily defined at 70% similarity) was prevalent in all three treatments . In addition, the distribution of RAPD clusters followed a log series model, regardless of the treatment applied, indicating that neither the microbial inoculant nor the fungicide treatment had caused a strong perturbation of the rhizobial population . When the P . fluorescens F113Rif and control treatments were compared using diversity indices, however, it appeared that the genetic diversity of rhizobia was significantly less in the inoculated treatment . The percentage of rhizobia sensitive to 2,4-diacetylphloroglucinol (Phl; the antimicrobial metabolite produced by P . fluorescens F113Rif) fluctuated according to field site heterogeneity, and treatments had no effect on this percentage . Yet, the proportion of Phl-sensitive isolates in the main RAPD cluster was lower in the P . fluorescens F113Rif treatment compared with the control, raising the possibility that the residual impact of the inoculant could have been partly mediated by production of Phl . This impact on the rhizobial population took place without affecting the functioning of the Rhizobium-clover symbiosis.

J Nat Prod, 2003 Jan, 66(1), 67 - 72
An efficient conversion of (3R,3'R,6'R)-lutein to (3R,3'S,6'R)-lutein (3'-epilutein) and (3R,3'R)-zeaxanthin; Khachik F; Two dietary carotenoids, (3R,3'R,6'R)-lutein (1) and (3R,3'R)-zeaxanthin (2), and their metabolite (3R,3'S,6'R)-lutein (3'-epilutein) (3) accumulate in human serum, milk, and ocular tissues . There is increasing evidence that compounds 1 and 2 play an important role in the prevention of age-related macular degeneration . Therefore, the availability of these carotenoids for metabolic studies and clinical trials is essential . Compound 1 is isolated from extracts of marigold flowers (Tagete erecta) and is commercially available, whereas 2 is only accessible by a lengthy total synthesis, and a viable method for synthesis of 3 has not yet been developed . This report describes an efficient conversion of technical grade 1 to 2 via 3 . Acid-catalyzed epimerization of 1 yields an equimolar mixture of diastereomers 1 and 3 . The mixture was separated by enzyme-mediated acylation with lipase AK from Pseudomonas fluorescens that preferentially esterified 3 and after alkaline hydrolysis yielded this carotenoid in 90% diastereomeric excess (de) . Compound 3 was also separated from 1 in 56-88% de by solvent extraction and low-temperature crystallization, Soxhlet extraction, or supercritical fluid extraction . Base-catalyzed isomerization of 3 gave 2 in excellent yield, providing a convenient alternative to the total synthesis of this important dietary carotenoid.

J Bacteriol, 2003 Feb, 185(3), 897 - 908
Plant lectin-like bacteriocin from a rhizosphere-colonizing Pseudomonas isolate; Parret AH et al.; Rhizosphere isolate Pseudomonas sp . strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P . putida GR12-2R3 . The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells . We isolated a TnMod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3 . A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells . The bacteriocin structural gene was identified by defining the minimal region required for expression in E . coli . This gene was designated llpA (lectin-like putidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants . LlpA is composed of two monocot mannose-binding lectin (MMBL) domains . Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified . A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family . Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains . In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence . Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.

J Bacteriol, 2003 Feb, 185(3), 860 - 9
2,5-dialkylresorcinol biosynthesis in Pseudomonas aurantiaca: novel head-to-head condensation of two fatty acid-derived precursors; Nowak-Thompson B et al.; 2-Hexyl-5-propylresorcinol is the predominant analog of several dialkylresorcinols produced by Pseudomonas aurantiaca (Pseudomonas fluorescens BL915) . We isolated and characterized three biosynthetic genes that encode an acyl carrier protein, a beta-ketoacyl-acyl carrier protein synthase III, and a protein of unknown function, all of which collectively allow heterologous production of 2-hexyl-5-propylresorcinol in Escherichia coli . Two regulatory genes exhibiting similarity to members of the AraC family of transcriptional regulators are also present in the identified gene cluster . Based on the deduced functions of the proteins encoded by the gene cluster and the observed incorporation of labeled carbons from octanoic acid into 2-hexyl-5-propylresorcinol, we propose that dialkylresorcinols are derived from medium-chain-length fatty acids by an unusual head-to-head condensation of beta-ketoacyl thioester intermediates . Genomic evidence suggests that there is a similar pathway for the biosynthesis of the flexirubin-type pigments in certain bacteria belonging to the order Cytophagales.

J Bacteriol, 2003 Feb, 185(3), 831 - 42
Elucidation of the Vibrio anguillarum genetic response to the potential fish probiont Pseudomonas fluorescens AH2, using RNA-arbitrarily primed PCR; Holmstrom K et al.; The antagonistic interaction between a potential fish probiont, Pseudomonas fluorescens strain AH2, and its target organism, Vibrio anguillarum, was investigated by studying the genetic response of the target organism when it was exposed to the antagonist . We compared the differential display of arbitrarily PCR-amplified gene transcripts in V . anguillarum serotype O1 when it was exposed to AH2 supernatant with the display of transcripts in nonexposed control cultures . Growth of V . anguillarum was immediately arrested when the organism was exposed to 50% (vol/vol) AH2 supernatant . A total of 10 potentially differentially expressed transcripts were identified . Among these we identified a gene homologous to rpoS that was induced in a dose-dependent manner when V . anguillarum was cultured in media supplemented with sterile filtered supernatant from AH2 . rpoS was also induced when growth was arrested with the iron chelator 2,2-dipyridyl . A chromosomal transcript homologous to vibE that participates in vibriobactin synthesis in Vibrio cholerae was also upregulated during AH2 exposure . This transcript could represent a functionally active gene in V . anguillarum involved in biosynthesis of anguibactin or another V . anguillarum siderophore . On the pJM1 plasmid of V . anguillarum serotype O1, a pseudogene designated open reading frame E (ORF E) that contains a frameshift mutation was previously identified . The gene homologous to vibE identified in this study, interestingly, also has significant homology to ORF E on the amino acid level and does not possess the frameshift mutation . Thus, the chromosomally encoded vibE homologue could fulfil the role of the inactive plasmid-encoded ORF E pseudogene . Addition of Fe(3+) to the system eliminated the growth arrest, and the genes homologous to rpoS and vibE were not induced . To our knowledge, this is the first study linking rpoS induction to iron starvation . Taken together, the results of this study suggest that a major part of the antagonistic property exhibited by strain AH2 is caused by the ability of siderophores in the supernatant to efficiently chelate iron, which results in instant iron deprivation of the pathogen V . anguillarum and complete growth arrest.

Curr Microbiol, 2003 Feb, 46(2), 131 - 40
Effect of plant growth-promoting Rhizobacteria and culture filtrate of Sclerotium rolfsii on phenolic and salicylic acid contents in chickpea (Cicer arietinum); Singh UP et al.; Two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens strain Pf4 and P . aeruginosa strain Pag, protected chickpea ( Cicer arietinum) plants from Sclerotium rolfsii infection when applied singly or in combination as seed treatment . Pag gave the best protection to the seedlings, applied either singly (mortality 16%) or in combination with Pf4 (mortality 17%) compared with 44% and 24% mortality in control and Pf4 treatment, respectively . The two PGPR strains induced the synthesis of specific phenolic acids, salicylic acid (SA), as well as total phenolics at different growth stages of chickpea seedlings with varied amount . The maximum amount of total phenolics was recorded in all the aerial parts of 4-week-old plants . Gallic, ferulic, chlorogenic, and cinnamic acids were the major phenolic acids detected in high-performance liquid chromatography (HPLC) analysis . Induction of such phenolic acids in the seedlings was observed up to 6 weeks in comparison with control . Salicylic acid (SA) was induced frequently during the first 3 weeks of growth only . Between the two strains, Pag was more effective in inducing phenolic acid synthesis applied either singly or in combination with strain Pf4 during the entire 6 weeks of growth of chickpea . In the presence of a culture filtrate of S . rolfsii, the two Pseudomonas strains induced more phenolic acids in treated than in non-treated and control plants . The occurrence of salicylic acid was frequent in the first 24 h, but infrequent at 48 and 96 h . Foliar spray of Pseudomonas strains also enhanced the phenolic acid content as well as total phenolics within 24 h of application . Gallic, chlorogenic, and cinnamic acids were consistently discerned in the treated leaves, whereas SA was absent even up to 96 h of application . Resistance in chickpea plants by Pseudomonas strains through induction of phenolic compounds as well as induced systemic resistance via SA-dependent pathway was evident.

Environ Health Perspect, 2003 Jan, 111(1), 85 - 92
Production of proinflammatory mediators by indoor air bacteria and fungal spores in mouse and human cell lines; Huttunen K et al.; We compared the inflammatory and cytotoxic responses caused by household mold and bacteria in human and mouse cell lines . We studied the fungi Aspergillus versicolor, Penicillium spinulosum, and Stachybotrys chartarum and the bacteria Bacillus cereus, Pseudomonas fluorescens, and Streptomyces californicus for their cytotoxicity and ability to stimulate the production of inflammatory mediators in mouse RAW264.7 and human 28SC macrophage cell lines and in the human A549 lung epithelial cell line in 24-hr exposure to 10(5), 10(6), and 10(7) microbes/mL . We studied time dependency by terminating the exposure to 10(6) microbes/mL after 3, 6, 12, 24, and 48 hr . We analyzed production of the cytokines tumor necrosis factor-alpha and interleukins 6 and 1ss (TNF-alpha, IL-6, IL-1ss, respectively) and measured nitric oxide production using the Griess method, expression of inducible NO-synthase with Western Blot analysis, and cytotoxicity with the MTT-test . All bacteria strongly induced the production of TNF-alpha, IL-6 and, to a lesser extent, the formation of IL-1ss in mouse macrophages . Only the spores of Str . californicus induced the production of NO and IL-6 in both human and mouse cells . In contrast, exposure to fungal strains did not markedly increase the production of NO or any cytokine in the studied cell lines except for Sta . chartarum, which increased IL-6 production somewhat in human lung epithelial cells . These microbes were less cytotoxic to human cells than to mouse cells . On the basis of equivalent numbers of bacteria and spores of fungi added to cell cultures, the overall potency to stimulate the production of proinflammatory mediators decreased in the order Ps . fluorescens > Str . californicus > B . cereus > Sta . chartarum > A . versicolor > P . spinulosum . These data suggest that bacteria in water-damaged buildings should also be considered as causative agents of adverse inflammatory effects.

Appl Environ Microbiol, 2003 Jan, 69(1), 686 - 90
Effect of stress on the ability of a phlA-based quantitative competitive PCR assay to monitor biocontrol strain Pseudomonas fluorescens CHA0; Rezzonico F et al.; A quantitative competitive PCR (QC-PCR) assay targeting the phlA gene of Pseudomonas fluorescens CHA0 was developed and tested in vitro . Statistically significant, positive correlations were found between QC-PCR and both CFU and total cell number when studying cells in log or stationary phase . The correlations disappeared when considering stressed cells.

Appl Environ Microbiol, 2003 Jan, 69(1), 419 - 26
Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase; Kamerbeek NM et al.; The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester . Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase . On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO . In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides . Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO . To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied . The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer . Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained {77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3} . Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%) . The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2113 - 20
Pseudomonas extremorientalis sp . nov., isolated from a drinking water reservoir; Ivanova EP et al.; On the basis of phenotypic and genotypic characteristics and 16S rDNA sequence analysis, a novel species belonging to the genus Pseudomonas sensu stricto was identified . The saprophytic, fluorescent bacterium, designated KMM 3447(T), was isolated from a drinking water reservoir near Vladivostok City, Russia . The novel organism was a Gram-negative, aerobic, rod-shaped bacterium that produced a cyclic depsipeptide with surface-active properties . It degraded casein, but did not degrade gelatin, starch, agar or Tween 80 . The bacterium was also haemolytic . Growth of the novel bacterium occurred between 4 and 35 degrees C . The predominant cellular fatty acids of the novel pseudomonad were C16:0, C16:1(n-7), C18:1(n-7) and C17.0 cyclo; branched fatty acids were only found in trace amounts . The G+C content of the novel bacterium was 61.0 mol% . 16S rDNA sequence analysis indicated that the novel bacterium had a clear affiliation with Pseudomonas fluorescens and species closely related to this recognized pseudomonad . DNA-DNA hybridization experiments showed that the novel bacterium bound at low levels (27-53%) with the DNA of the type strains of its nearest phylogenetic relatives, namely Pseudomonas tolaasii, Pseudomonas veronii, Pseudomonas orientalis and Pseudomonas rhodesiae, indicating that the novel bacterium represented a novel species within the genus Pseudomonas, for which the name Pseudomonas extremorientalis is proposed; the type strain is KMM 3447(T) (= LMG 19695(T)).

Biosci Biotechnol Biochem, 2002 Nov, 66(11), 2471 - 3
Siderophore production and induction of iron-regulated proteins by a microorganism from rhizosphere of barley; Terano H et al.; This study provides new information on the Fe uptake system capable of supporting growth of the organism . Pseudomonas fluorescens isolated from the rhizosphere of barley, a gramineous plant, produced a siderophore under iron-limiting conditions . Its chemical structure was identified as pyochelin, on the basis of 1H and 13C NMR data of a stable methyl ester derivative . The same iron-limiting conditions induced a new set of outer membrane proteins (75 and 55 kDa), consistent with a siderophore-mediated iron-uptake system.

Biochim Biophys Acta, 2003 Jan 2, 1619(1), 70 - 6
Aluminum detoxification in Pseudomonas fluorescens is mediated by oxalate and phosphatidylethanolamine; Hamel R et al.; 13C NMR studies with aluminum (Al)-stressed Pseudomonas fluorescens revealed that the trivalent metal was secreted in association with oxalate and phosphatidylethanolamine (PE) . These moieties were observed in the insoluble pellet obtained upon incubation of these resting cells in the presence of either Al-citrate or citrate . This extrusion process was concomitant with the utilization of either of these tricarboxylic acids as a substrate . While only minimal amounts of Al were secreted in the presence of such carbon source as glucose, succinate or oxaloacetate, oxalate did permit the efflux of Al . Neither alpha-ketoglutarate nor ethylenediaminetetraacetic acid (EDTA) was effective in dislocating Al from the cells . The elimination of Al from the cells did not appear to be affected by p-dinitrophenol (DNP) or dicyclohexylcarbodiimide (DCCD) or azide, but was sensitive to temperature, pH and cerulenin, an inhibitor of lipid synthesis . Thus, it appears that P . fluorescens detoxifies Al via its extrusion in association with oxalate and PE in a process that apparently does not necessitate the direct utilization of energy.

J Food Prot, 2002 Dec, 65(12), 1924 - 9
Effect of dissolved carbon dioxide on thermal inactivation of microorganisms in milk; Loss CR et al.; Postpasteurization addition of CO2 inhibits growth of certain microorganisms in dairy products, but few workers have investigated the effect of CO2 on the thermal inactivation of microorganisms during pasteurization . Concentrations of CO2 ranging from 44 to 58 mM added to raw whole milk significantly (P < 0.05) reduced the number of surviving standard plate count (SPC) organisms in milk heated over the range of 67 to 93 degrees C . A decrease in thermal survival rates (D-values) for Pseudomonas fluorescens R1-232 and Bacillus cereus ATCC 14579 spores in milk was positively correlated with CO2 concentrations (1 to 36 mM) . D(50 degrees C)-values for P . fluorescens significantly decreased (P < 0.05) in a linear fashion from 14.4 to 7.2 min . D(89 degrees C)-values for B . cereus spores were significantly (P < 0.05) decreased from 5.56 min in control milk to 5.29 min in milk containing 33 mM CO2 . The Weibull function was used as a model to describe the thermal inactivation of P . fluorescens, B . cereus spores, and SPC organisms in raw milk . Nonlinear parameters for the Weibull function were estimated, and survival data fitted to this model had higher R2 values than when fitted to the linear model, further providing support that the thermal inactivation of bacteria does not always follow first-order reaction rate kinetics . These results suggest that CO2 could be used as a processing aid to enhance microbial inactivation during pasteurization.

J Exp Bot, 2003 Jan, 54(381), 325 - 34
Biosensor reporting of root exudation from Hordeum vulgare in relation to shoot nitrate concentration; Darwent MJ et al.; The aim of this study was to determine the relationship between shoot nitrate concentration, mediated by nitrate supply to roots, and root exudation from Hordeum vulgare . Plants were grown for 14 d in C-free sand microcosms, supplied with nutrient solution containing 2 mM nitrate . After this period, three treatments were applied for a further 14 d: (A) continued supply with 2 mM nitrate (zero boost), (B) supply with 10 mM nitrate (low boost), and (C) supply with 20 mM nitrate (high boost) . At the end of the treatment period, a bacterial biosensor (Pseudomonas fluorescens 10586 pUCD607, marked with the lux CDABE genes for bioluminescence) was applied to the microcosms to report on C-substrate availability, as a consequence of root exudation . The nitrate boost treatments significantly affected shoot nitrate concentrations, in the order C>B>A . In treatments receiving a nitrate boost (B, C), increased shoot nitrate concentration was correlated with increased plant biomass, reduced root length, reduced number of root tips, and increased mean root diameter, relative to the no boost treatment (A) . Imaging of biosensor bioluminescence (proportional to metabolic activity in response to availability of root exudates) indicated that root exudation increased with decreasing shoot nitrate concentration . Biosensor reporting of root C-flow indicated that exudation was greater from root tip regions than from the whole root, but that specific exudation rates for all sites were unaffected by treatments . Total root exudation across treatments was found to be closely correlated with total root length, indicating that increased root exudation, per unit root biomass, with decreasing nitrate supply was associated with altered root morphology, as a consequence of systemic plant responses to internal N-status.

Can J Microbiol, 2002 Oct, 48(10), 940 - 4
Effective dose of a microbial inoculant is one to four cells in the rhizosphere; Normander B et al.; A single-cell approach for studying the growth potential and the establishment of bacteria in the barley phytosphere is presented, using Pseudomonas fluorescens strain with the capability for biological control . The incidence of growth of one to four bacterial cells dispersed to the young rhizosphere approximated to 100%, and specific growth rate averaged 0.05 . Net growth occurred for cells added to the rhizosphere at densities between 1 and 100,000 cells, while at higher densities population sizes declined, but always approached 10(5)-10(6) cells per rhizosphere . No net growth was observed in bulk soil, and cells died in the phyllosphere . Our results showed that bacterial establishment was more related to the availability of microhabitats supporting growth, than related to the number of bacteria released.

Biotechnol Bioeng, 2003 Feb 5, 81(3), 370 - 8
Kinetic analysis of bacterial bioluminescence; Kelly CJ et al.; Bioluminescence from the lux-based bacterial reporter Pseudomonas fluorescens HK44 was experimentally investigated under growth substrate-rich and limiting conditions in batch, continuous stirred tank (CSTR), and turbidostat reactors . A mechanistically based, mathematical model was developed to describe bioluminescence based on 1) production and decay of catalytic enzymes, and 2) reactant cofactor availability . In the model, bioluminescence was a function of inducer, growth substrate, and biomass concentration . A saturational dependence on growth substrate concentration accommodated dependence on cofactor availability and inducer concentration to accommodate enzyme production was incorporated in the model . Under growth substrate and inducer limiting conditions in the batch reactor and CSTR, bioluminescence was found to decrease in response to cellular energy limitations . The effective lux system enzyme decay rate was determined in independent measurements to be 0.35 hr(-1) and the model captured most of the bioluminescent behavior, except at long growth times and high cell density .

Nature, 2002 Dec 5, 420(6915), 496 - 9
The role of parasites in sympatric and allopatric host diversification; Buckling A et al.; Exploiters (parasites and predators) are thought to play a significant role in diversification, and ultimately speciation, of their hosts or prey . Exploiters may drive sympatric (within-population) diversification if there are a variety of exploiter-resistance strategies or fitness costs associated with exploiter resistance . Exploiters may also drive allopatric (between-population) diversification by creating different selection pressures and increasing the rate of random divergence . We examined the effect of a virulent viral parasite (phage) on the diversification of the bacterium Pseudomonas fluorescens in spatially structured microcosms . Here we show that in the absence of phages, bacteria rapidly diversified into spatial niche specialists with similar patterns of diversity across replicate populations . In the presence of phages, sympatric diversity was greatly reduced, as a result of phage-imposed reductions in host density decreasing competition for resources . In contrast, allopatric diversity was greatly increased as a result of phage-imposed selection for resistance, which caused populations to follow divergent evolutionary trajectories . These results show that exploiters can drive diversification between populations, but may inhibit diversification within populations by opposing diversifying selection that arises from resource competition.

J Appl Microbiol, 2002, 93(6), 1065 - 74
Impact of biocontrol strain Pseudomonas fluorescens CHA0 on rhizosphere bacteria isolated from barley (Hordeum vulgare L.) with special reference to Cytophaga-like bacteria; Johansen JE et al.; AIMS: To assess the impact of the biocontrol strain Pseudomonas fluorescens CHA0 on a collection of barley rhizosphere bacteria using an agar plate inhibition assay and a plant microcosm, focusing on a CHA0-sensitive member of the Cytophaga-like bacteria (CLB) . METHODS AND RESULTS: The effect of strain CHA0 on a collection of barley rhizosphere bacteria, in particular CLB and fluorescent pseudomonads sampled during a growth season, was assessed by a growth inhibition assay . On average, 85% of the bacteria were sensitive in the May sample, while the effect was reduced to around 68% in the July and August samples . In the May sample, around 95% of the CLB and around 45% of the fluorescent pseudomonads were sensitive to strain CHA0 . The proportion of CHA0-sensitive CLB and fluorescent pseudomonad isolates decreased during the plant growth season, i.e . in the July and August samples . A particularly sensitive CLB isolate, CLB23, was selected, exposed to strain CHA0 (wild type) and its genetically modified derivatives in the rhizosphere of barley grown in gnotobiotic soil microcosms . Two dry-stress periods were imposed during the experiment . Derivatives of strain CHA0 included antibiotic or exopolysaccharide (EPS) overproducing strains and a dry-stress-sensitive mutant . Despite their inhibitory activity against CLB23 in vitro, neither wild-type strain CHA0, nor any of its derivatives, had a major effect on culturable and total cell numbers of CLB23 during the 23-day microcosm experiment . Populations of all inoculants declined during the two dry-stress periods, with soil water contents below 5% and plants reaching the wilting point, but they recovered after re-wetting the soil . Survival of the dry-stress-sensitive mutant of CHA0 was most affected by the dry periods; however, this did not result in an increased population density of CLB23 . CONCLUSIONS: CLB comprise a large fraction of barley rhizosphere bacteria that are sensitive to the biocontrol pseudomonad CHA0 in vitro . However, in plant microcosm experiments with varying soil humidity conditions, CHA0 or its derivatives had no major impact on the survival of the highly sensitive CLB strain, CLB23, during two dry-stress periods and a re-wetting period; all co-existed well in the rhizosphere of barley plants . SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate a lack of interaction between the biocontrol pseudomonad CHA0 and a sensitive CLB when the complexity increases from agar plate assays to plant microcosm experiments . This suggests the occurrence of low levels of antibiotic production and/or that the two bacterial genera occupy different niches in the rhizosphere.

Mikrobiologiia, 2002 Sep-Oct, 71(5), 629 - 34
{Extracellular protease as a reversible adhesion regulator in Pseudomonas fluorescens}; Nikolaev IuA et al.; The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2-3 h of growth . Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h-1, the fraction of proteins in cells being almost the same (60-70%) . The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture . The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium . After 3-5 h of growth, the concentration of extracellular proteins decreased to the control level . Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells . The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.

Mikrobiologiia, 2002 Sep-Oct, 71(5), 619 - 28
{Growth and adhesion of Pseudomonas fluorescens in a batch culture: a kinetic analysis of the action of extracellular antiadhesins}; Panikov NS et al.; The work varifies 6 hypothetical models simulating the growth, respiration, and adhesion of cells to the walls of the cultivation flask . All the models postulate the synthesis of antiadhesins (AAs), i.e., extracellular metabolites decreasing the degree of cell adhesion . The models have the following distinguishing features: (model 1) the blocking of sorption centers on the glass walls by antiadhesins (the competitive inhibition of adhesion); (model 2) the noncompetitive inhibition of adhesion; (model 3) the accelerated release of bound cells; (model 4) a combination of models 1 and 3; (model 5) a combination of models 1 and 3 with a delay; (model 6) a combined action of two AAs, one of which, AA1, inhibits cell adhesion, and the other, AA2 (its synthesis is induced when the concentration of AA1 reaches a threshold level), stimulates the detachment of bound cells . Model 6 fits the relevant experimental data best . The delay effect is relatively small . The sigmoid character of the curve showing cell adhesion as a function of the antiadhesin concentration implies the existence of a strong cooperative effect in the adhesion inhibition . The models proposed satisfactorily simulate the growth, respiration, and adhesion of cells and AA synthesis in a batch bacterial culture grown either in a fresh nutrient medium or in the medium supplemented with the filtrate of a mature culture of the same species.

Infect Immun, 2002 Dec, 70(12), 6567 - 75
Pseudomonas fluorescens encodes the Crohn's disease-associated I2 sequence and T-cell superantigen; Wei B et al.; Commensal bacteria have emerged as an important disease factor in human Crohn's disease (CD) and murine inflammatory bowel disease (IBD) models . We recently isolated I2, a novel gene segment of microbial origin that is associated with human CD and that encodes a T-cell superantigen . To identify the I2 microorganism, BLAST analysis was used to identify a microbial homologue, PA2885, a novel open reading frame (ORF) in the Pseudomonas aeruginosa genome . PCR and Southern analysis identified Pseudomonas fluorescens as the originating species of I2, with homologues detectable in 3 of 13 other Pseudomonas species . Genomic cloning disclosed a locus containing the full-length I2 gene (pfiT) and three other orthologous genes, including a homologue of the pbrA/pvdS iron response gene . CD4(+) T-cell responses to recombinant proteins were potent for I2 and pfiT, but modest for PA2885 . pfiT has several features of a virulence factor: association with an iron-response locus, restricted species distribution, and T-cell superantigen bioactivity . These findings suggest roles for pfiT and P . fluorescens in the pathogenesis of Crohn's disease.

Res Microbiol, 2002 Oct, 153(8), 527 - 36
Integration host factor is essential for the optimal expression of the styABCD operon in Pseudomonas fluorescens ST; Santos PM et al.; The StyS/StyR two-component regulatory system of Pseudomonas fluorescens ST controls the expression of the styABCD operon coding for the styrene degradation upper pathway . In a previous work we showed that the promoter of the catabolic operon (PstyA) is induced by styrene and repressed to differing extents by organic acids or carbohydrates . In order to study the mechanisms controlling the expression of this operon, we performed a functional analysis on 5' deletions of PstyA by the use of a promoter-probe system . These studies demonstrated that a palindromic region (sty box), located from nucleotides -52 to -37 with respect to the transcriptional start point is essential for PstyA activity . Moreover, additional regulatory regions involved in the modulation of PstyA activity were found along the promoter sequence . In particular, deletion of a putative StyR binding site, homologous to the 3' half of the sty box and located upstream of this box, resulted in 65% reduction of the induction level of the reporter gene . Additionally, we performed bandshift assays with a DNA probe corresponding to PstyA and protein crude extracts from P . fluorescens ST, using specific DNA fragments as competitors . In these experiments we demonstrated that IHF binds an AT-rich region located upstream of the sty box . On the basis of this finding, coupled with the results obtained with PstyA functional analysis, we suggest that the role of the IHF-mediated DNA bend is to bring closer, in an overlapping position, the upstream StyR putative binding site and the downstream sty box, and that the formed complex enhances transcription.

Mol Genet Genomics, 2002 Nov, 268(3), 387 - 96 Epub 2002 Oct 11.
Genetic analysis of the biosynthesis of the pyrrole and carbamoyl moieties of coumermycin A1 and novobiocin; Xu H et al.; The aminocoumarin antibiotic coumermycin A(1) contains a central and two terminal pyrrole moieties . The coumermycin gene cluster in Streptomyces rishiriensis contains three genes (couN3, couN4 and couN5) that show sequence similarity to genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens and of undecylprodiginine in S . coelicolor . The gene couN3, which codes for a putative L-prolyl-S-PCP dehydrogenase, and the gene couN4, which encodes a putative L-prolyl-AMP ligase, were disrupted using in-frame deletion and insertional inactivation, respectively . HPLC analysis of culture extracts showed that formation of the two terminal pyrrole moieties was abolished in the couN3 (-) und couN4 (-) mutants . The mutants accumulated coumermycin D, which contains only the central pyrrole moiety . This result not only confirmed the involvement of couN3 and couN4 in the biosynthesis of the terminal pyrrole-2-carboxylic acid moieties of coumermycin A(1), but also indicated, for the first time, that the central 3-methylpyrrole-2,4-dicarboxylic acid unit of the coumermycins is formed by a biosynthetic pathway that differs from that used to assemble the terminal pyrrole moieties . novN, a putative carbamoyl transferase gene from the gene cluster for novobiocin biosynthesis in S . spheroides was expressed in the couN3 (-) mutant . This led to the formation of bis-carbamoylated coumermycin D, a novel compound of the coumermycin series.

Carbohydr Res, 2002 Nov 19, 337(21-23), 2365 - 70
Somatic antigens of pseudomonads: structure of the O-specific polysaccharide of Pseudomonas fluorescens IMV 2366 (biovar C); Zatonsky GV et al.; The O-specific polysaccharide of P . fluorescens IMV 2366 was studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D gsCOSY, TOCSY, gsNOESY, H-detected 1H,(13)C gsHSQC, HMQC-TOCSY, and gsHMBC experiments . The polysaccharide contains L-rhamnose, 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) and 3-acylamido-3,6-dideoxy-D-glucose (D-Qui3NAcyl, where Acyl is 3-hydroxy-2,3-dimethyl-5-oxoprolyl) . The structure 1 of the polysaccharide was found to be similar to the structure 2 of a 6-deoxy-L-talose (L-6dTal)-containing O-specific polysaccharide of a non-classified P . fluorescens strain, 361, studied earlier {Khomenko, V . A.; Naberezhnykh, G . A.; Isakov, V . V.; Solov'eva, T . F.; Ovodov, Y . S.; Knirel, Y . A.; Vinogradov, E . V . Bioorg . Khim . 1986, 12, 1641-1648; Naberezhnykh, G . A.; Khomenko, V . A.; Isakov, V . V., El'kin, Y . N.; Solov'eva, T . F.; Ovodov, Y . S . Bioorg . Khim . 1987, 13, 1428-1429} . --> 2)-beta-D-Quip3NAcyl-(1 --> 3)-alpha-L-Rhap-(1 --> 3)-alpha-D-FucpNAc-(1 --> 1 . --> 4)-beta-D-Quip3NAcyl-(1 --> 3)-alpha-L-6dTalp4Ac-(1 --> 3)-alpha-D-FucpNAc-(1 -->2 .

Proc R Soc Lond B Biol Sci, 2002 Nov 7, 269(1506), 2277 - 83
Mechanisms linking diversity, productivity and invasibility in experimental bacterial communities; Hodgson DJ et al.; Decreasing species diversity is thought to both reduce community productivity and increase invasibility to other species . However, it remains unclear whether identical mechanisms drive both diversity-productivity and diversity-invasibility relationships . We found a positive diversity-productivity relationship and negative diversity-invasibility and productivity-invasibility relationships using microcosm communities constructed from spatial niche specialist genotypes of the bacterium Pseudomonas fluorescens . The primary mechanism driving these relationships was a dominance (or selection) effect: more diverse communities were more likely to contain the most productive and least invasible type . Statistical elimination of the dominance effect greatly weakened the diversity-invasibility relationship and eliminated the diversity-productivity relationship, but also revealed the operation of additional mechanisms (niche complementarity, positive and negative interactions) for particular combinations of niche specialists . However, these mechanisms differed for invasibility and productivity responses, resulting in the invasibility-productivity relationship changing from strongly negative to weakly positive . In the absence of the dominance effect, which may be an experimental artefact, decreasing diversity can have unexpected or no effects on ecosystem properties.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2001, 66(2a), 95 - 101
Antagonistic effects of several bacteria on Verticillium dahliae the causal agent of cotton wilt; Tehrani AS et al.; Experiments were carried out with 89 bacterial isolates that were collected from cotton rhizosphere in Gorgan province . The antagonistic effects of bacterial isolates on Verticillium dahliae Klebahn were studied using dual culture test . Five highly effective isolates were selected from these antagonists for subsequent studies . According to the biochemical, physiological and morphological tests, isolates 2020 and 3 were identified as Pseudomonas fluorescens and isolate 204, 202 and 309 as Bacillus spp . These isolates were used to investigate their antagonistic mechanisms in vitro and their effects on cotton growth in vivo . Inhibition of V . dahliae by volatile metabolites and antibiotics was studied as described by Fiddamen (1994) and Kraus (1990) . Production of hydrogen cyanide was studied qualitatively, using HCN-indicator paper of Castric and Castric (1983) . Isolates 204, 202 and 309 inhibited the mycelial growth of the fungus through production of volatile metabolites . Isolates 2020 and 3 produced antibiotic as well as volatile metabolities that inhibited mycelial growth of V . dahliae . They both produced hydrogen cyanide . After four months of greenhouse study, the application of antagonistic bacteria had different effects on growth of cotton plants . Bacterial treatment in soil had better effects on plant growth than that of bacterial seed treatment . In soil treatments containing infested and non-infested soil with V . dahliae, isolates 2020 and 3 caused an increase in plant height in comparison with those in infested and non-infested controls . In non-infested soil, application of isolates 2020, 3 and 202 increased root length and dry weight of cotton plant, but in soil infested with the fungus, only isolate 202 increased root length . Isolate 2020 increased plant dry weight . In conclusion, isolates 2020 and 3 belonging to P . fluorescens and isolate 202 pertaining to genus Bacillus had the greatest effect on increasing the cotton growth.

Carbohydr Res, 2002 Oct 8, 337(18), 1615 - 21
Regioselective enzymatic hydrolysis of acetylated pyranoses and pyranosides using immobilised lipases . An easy chemoenzymatic synthesis of alpha- and beta-D-glucopyranose acetates bearing a free secondary C-4 hydroxyl group; Terreni M et al.; Protected sugars with only one free hydroxyl group are useful building blocks for the synthesis of a large number of glycoderivatives . In order to avoid the problems of the classical chemical synthesis, we studied the regioselective enzymatic hydrolysis of different fully acetylated glycopyranoses and glycopyranosides . The main challenge was to obtain the hydrolysis of the substrates in only one position, with high regioselectivity, while avoiding any further hydrolysis towards partially acetylated sugars . Candida rugosa (CRL) and Pseudomonas fluorescens (PFL) lipases (EC 3.1.1.3) immobilised on octyl agarose afforded regioselective hydrolysis only in the 6- and 1-positions, respectively . Furthermore, a new one-pot chemoenzymatic approach has been developed in order to obtain alpha- and beta-protected glucopyranoses bearing a free secondary C-4 hydroxyl group . For instance, 1,2,3,6-tetra-O-acetyl-alpha-D-glucopyranose was easily synthesised in good overall yield (70%) starting from 1,2,3,4,6-penta-O-acetyl-alpha-D-glucopyranose by regioselective enzymatic hydrolysis in the 6-position, catalysed by CRL, followed by a temperature- and pH-controlled acyl migration.

Eur J Biochem, 2002 Nov, 269(22), 5391 - 405
Galactosyl-mimodye ligands for Pseudomonas fluorescens beta-galactose dehydrogenase; Mazitsos CF et al.; Protein molecular modelling and ligand docking were employed for the design of anthraquinone galactosyl-biomimetic dye ligands (galactosyl-mimodyes) for the target enzyme galactose dehydrogenase (GaDH) . Using appropriate modelling methodology, a GaDH model was build based on a glucose-fructose oxidoreductase (GFO) protein template . Subsequent computational analysis predicted chimaeric mimodye-ligands comprising a NAD-pseudomimetic moiety (anthraquinone diaminobenzosulfonic acid) and a galactosyl-mimetic moiety (2-amino-2-deoxygalactose or shikimic acid) bearing an aliphatic 'linker' molecule . In addition, the designed mimodye ligands had an appropriate in length and chemical nature 'spacer' molecule via which they can be attached onto a chromatographic support without steric clashes upon interaction with GaDH . Following their synthesis, purification and analysis, the ligands were immobilized to agarose . The respective affinity adsorbents, compared to other conventional adsorbents, were shown to be superior affinity chromatography materials for the target enzyme, Pseudomonas fluorescensbeta-galactose dehydrogenase . In addition, these mimodye affinity adsorbents displayed good selectivity, binding low amounts of enzymes other than GaDH . Further immobilized dye-ligands, comprising different linker and/or spacer molecules, or not having a biomimetic moiety, had inferior chromatographic behavior . Therefore, these new mimodyes suggested by computational analysis, are candidates for application in affinity labeling and structural studies as well as for purification of galactose dehydrogenase.

Mol Plant Microbe Interact, 2002 Nov, 15(11), 1173 - 80
Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens; de Weert S et al.; Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens . To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P . fluorescens strains . After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil . The differences were significant on all root parts and increased from root base to root tip . Significant differences at the root tip could already be detected after 2 to 3 days . These experiments show that chemotaxis is an important competitive colonization trait . The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components . A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars . Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P . fluorescens WCS365 cells in the tomato rhizosphere.

Ann Chim, 2002 Sep, 92(9), 847 - 54
A new microbial assay for the toxicity detection of contaminated soils; Guerra R et al.; A comparative study to detect toxicity prior to bioremediation treatment was set in order to investigate dehydrogenase activity inhibition of a common soil bacterium caused by soil contaminated with Cu, Pb, and As . A spectrophotometric test with Pseudomonas fluorescens strain ATCC 13525 utilising the 2,3,5-triphenyl tetrazolium chloride (TTC) reduction by microbial dehydrogenase has been adapted for this purpose . Soil samples are incubated for 48 hours at 30 +/- 1 degrees C in 18-ml tubes in the presence of TTC as an artificial electron-acceptor . The reduced TTC forms a reddish colour substance named triphenyl formazan (TPF), which can be extracted from the microbial cells and measured colorimetrically . The rapid response of biological activity in microorganisms and the reported sensitivity to the toxicants in the contaminated samples are reflected by the TTC reduction method, which is a sensitive tool for toxicity screening of contaminated sites, routine monitoring of bioremediation processes, as well as for feasibility studies of bioremediation treatments, in order to assess whether a specific pollutant or any other substance at a site location could inhibit the microbiological processes.

J Microbiol Methods, 2003 Jan, 52(1), 47 - 58
Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria; Hatzinger PB et al.; A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC) . In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) . To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3'-{1-(phenylamino)-carbonyl}-3,4-tetrazolium}-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated . Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays . For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude . A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation . Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation . However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation . This strain would be considered nonviable based on traditional tetrazolium salt reduction assays . The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 121 - 6
Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins; Rebiere-Huet J et al.; Bacterial adherence is a complex phenomenon involving specific interactions between receptors, including matricial fibronectin, and bacterial ligands . We show here that fibronectin and outer membrane proteins of Pseudomonas fluorescens were able to inhibit adherence of P . fluorescens to fibronectin-coated wells . We identified at least six fibronectin-binding proteins with molecular masses of 70, 55, 44, 37, 32 and 28 kDa . The presence of native (32 kDa) and heat-modified forms (37 kDa) of OprF was revealed by immuno-analysis and the 44-kDa band was composed of three proteins, their N-terminal sequences showing homologies with Pseudomonas aeruginosa porins (OprD, OprE1 and OprE3).

Lett Appl Microbiol, 2002, 35(5), 380 - 4
Control of pyrimidine synthesis in Pseudomonas fragi; West TP; AIMS: To study the regulation of the de novo pyrimidine biosynthetic enzymes in the food-spoilage agent Pseudomonas fragi ATCC 4973 . METHODS AND RESULTS: The de novo pyrimidine biosynthetic enzymes were measured in extracts of Ps . fragi ATCC 4973 cells and of cells from auxotrophs deficient for dihydroorotase or OMP decarboxylase activity . Pyrimidine biosynthetic enzyme activities in ATCC 4973 were influenced by pyrimidine supplementation to the culture medium . The pyrimidine limitation of each auxotroph elevated the de novo enzyme activities, indicating that this pathway may be repressible by a pyrimidine-related compound . Aspartate transcarbamoylase activity in ATCC 4973 was inhibited in vitro by pyrophosphate and purine or pyrimidine nucleotides . CONCLUSIONS: Pyrimidine synthesis in Ps . fragi appeared to be controlled at the transcriptional level and at the level of activity for aspartate transcarbamoylase . Its transcriptional regulation seemed to be more highly controlled than what was observed in the closely related species Pseudomonas putida and Pseudomonas fluorescens . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that pyrimidine synthesis is regulated in Ps . fragi . This could prove useful to future studies examining its biological control and its taxonomic assignment.

Environ Sci Technol, 2002 Oct 15, 36(20), 4334 - 45
Utilization and transformation of aquatic humic substances by autochthonous microorganisms; Hertkorn N et al.; Aquatic humic substances (HS) from a bog lake water, a riverwater, and a groundwater were isolated after enrichment on XAD 8 columns and added to a Czapek-Dox nutrient broth which was used either in full strength or without glucose and/or NaNO3 . The individual flasks were inoculated with natural microbial populations of corresponding water samples or with a Pseudomonas fluorescens strain isolated from groundwater . The presence of HS resulted in an increase of bacterial numbers in nearly all cultures incubated for 3 weeks at 25 degrees C on a shaker . HS reisolated from cultures without glucose or NaNO3 showed no or only minor quantitative differences as compared to those from sterile controls . In full strength nutrient broth up to 27% of HS were utilized . Data obtained by spectroscopic methods (UV/vis/FTIR) and elemental analysis indicated a decrease in particle size and a loss in aromaticity and aliphatic carbon in HS reisolated from the microbial cultures . Simultaneously an increase in the N content of HS was observed, which probably originated from some constituents of microbial biomass such as proteins and amino sugars . The NMR data also documented that significant transformations of HS occurred in the individual microbial cultures . After incubation, increased amounts of aromatic acids were detected in some liquid media and residual HS by GC/MS or capillary electrophoresis . 1H NMR spectroscopy was less effective in indicating structural differences in the HS than 13C NMR but revealed considerable detail of the microbial degradation of riverine HS, when limited sample was available . The newly developed NMR increment analysis provided substantial detail of aromatic structures in a microbially altered HS . The microbial degradation of HS strongly depended on the composition of the HS, the species selection of the microorganisms, and to a lesser extent on the culture conditions . For any series of identical inoculum and HS, full broth media initiated the most extensive alteration of HS.

Nucleic Acids Res . 2002 Oct 1;30(19):e98.
PfoI, a unique type II restriction endonuclease that recognises the sequence 5'-T downward arrow CCNGGA-3'; Gaigalas M et al.; A new type II restriction endonuclease designated PfoI has been partially purified from Pseudomonas fluorescens biovar 126 . PfoI recognises the interrupted hexanucleotide palindromic sequence 5'-T downward arrow CCNGGA-3' and cleaves DNA to produce protruding pentanucleotide 5'-ends.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1497 - 503
Pseudomonas grimontii sp . nov; Baida N et al.; The vernacular name 'fluorescent Pseudomonas group 97-514' was coined for a group of 43 strains isolated from two French natural mineral waters . All these strains were gram-negative, rod-shaped and motile by means of a single polar flagellum . They produced fluorescent pigment (pyoverdin) on King B medium, catalase and cytochrome oxidase . They were capable of respiratory but not fermentative metabolism . They were not able to accumulate poly-beta-hydroxybutyrate and possessed an arginine dihydrolase system . DNA-DNA relatedness studies (S1 nuclease method) showed that the 43 strains of 'fluorescent Pseudomonas group 97-514' formed a genetically homogeneous group (DNA-DNA relatedness ranged from 70 to 100%) . A total of 76 strains representing well-known or partially characterized species of the genus Pseudomonas sensu stricto had 7-56% DNA hybridization with strain CFML 97-514T . The highest DNA binding values were found with Pseudomonas veronii CIP 104663T (52%), Pseudomonas rhodesiae CIP 104664T (56%), Pseudomonas marginalis ATCC 10844T (56%), Pseudomonas gessardii CIP 105469T (53%) and Pseudomonas cedrella CIP 105541T (52%) . Their unrelatedness was confirmed by deltaTm values greater than 7 degrees C . On the basis of the results of phenotypic and DNA-DNA hybridization studies, a novel Pseudomonas species, Pseudomonas grimontii sp . nov., is proposed for the 43 strains of 'fluorescent Pseudomonas group 97-514' . The type strain is strain CFML 97-514T (= CIP 106645T = ATCC BAA-140T) . The G+C content of the DNA of the type strain was 58 mol% . A comparison of the complete 16S rRNA gene sequence of the type strain CFML 97-514T and the sequence of other strains of the genus Pseudomonas revealed that the novel species fell within the 'Pseudomonas fluorescens intrageneric cluster' . Members of P . grimontii grew at 4 degrees C but not at 41 degrees C . They were able to use D-xylose, alpha-L-rhamnose, alpha-aminobutyrate, meso-erythritol and itaconate as sole sources of carbon and energy and formed levan from sucrose . Strains do not possess lecithinase or Tween esterase activities . The clinical significance of P . grimontii is unknown.

Mol Microbiol, 2002 Sep, 45(6), 1673 - 85
Identification of new, conserved, non-ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine; Mossialos D et al.; Pyoverdines, the main siderophores of fluorescent pseudomonads, contain a peptide moiety, different for each pyoverdine, and an identical chromophore . While it has been shown that non-ribosomal peptide synthetases (NRPSs) are involved in the biosynthesis of the peptide chain of pyoverdines, this was not demonstrated for the biosynthesis of the chromo-phore part . We found that PvsA, from Pseudomonas fluorescens ATCC 17400, and PvdL (PA2424), from Pseudomonas aeruginosa are similar NRPSs and functional homologues, necessary for the production of pyoverdine . Transcriptional lacZ fusions showed that pvdL is co-transcribed with the upstream PA2425 gene, encoding a putative thioesterase, and is iron-regulated via PvdS . Similarly, RT-PCR analysis revealed that expression of pvsA is repressed by iron . Analysis of the adenylation domains of PvsA, PvdL and their homologues, revealed that their N-terminus starts with an acyl-CoA ligase module, followed by three amino acid activation domains . Computer modelling of these domains suggests that PvsA in P . fluorescens and PvdL in P . aeruginosa are orthologues involved in the biosynthesis of the pyoverdine chromophore.

Appl Environ Microbiol, 2002 Oct, 68(10), 5170 - 6
Identification of differences in genome content among phlD-positive Pseudomonas fluorescens strains by using PCR-based subtractive hybridization; Mavrodi DV et al.; Certain 2,4-diacetylphloroglucinol-producing strains of Pseudomonas fluorescens colonize roots and suppress soilborne diseases more effectively than others from which they are otherwise phenotypically almost indistinguishable . We recovered DNA fragments present in the superior colonizer P . fluorescens Q8r1-96 but not in the less rhizosphere-competent strain Q2-87 . Of the open reading frames in 32 independent Q8r1-96-specific clones, 1 was similar to colicin M from Escherichia coli, 3 resembled known regulatory proteins, and 28 had no significant match with sequences of known function . Seven clones hybridized preferentially to DNA from strains with superior rhizosphere competence, and sequences in two others were highly expressed in vitro and in the rhizosphere.

Sens Actuators B Chem, 2002 Jun 20, 85(1-2), 179 - 85
Integrated CMOS photodetectors and signal processing for very low-level chemical sensing with the bioluminescent bioreporter integrated circuit; Bolton EK et al.; We report an integrated CMOS microluminometer optimized for the detection of low-level bioluminescence as part of the bioluminescent bioreporter integrated circuit (BBIC) . This microluminometer improves on previous devices through careful management of the sub-femtoampere currents, both signal and leakage, that flow in the front-end processing circuitry . In particular, the photodiode is operated with a reverse bias of only a few mV, requiring special attention to the reset circuitry of the current-to-frequency converter (CFC) that forms the front-end circuit . We report a sub-femtoampere leakage current and a minimum detectable signal (MDS) of 0.15 fA (1510 s integration time) using a room temperature 1.47 mm2 CMOS photodiode . This microluminometer can detect luminescence from as few as 5000 fully induced Pseudomonas fluorescens 5RL bacterial cells . c2002 Elsevier Science B.V . All rights reserved.

Bioresour Technol, 2002 Nov, 85(2), 213 - 5
Effects of root-dip treatment with certain phosphate solubilizing microorganisms on the fusarial wilt of tomato; Khan MR et al.; Root-dip application of Bacillus subtilis, Pseudomonas fluorescens, Aspergillus awamori, Aspergillus niger and Penicillium digitatum resulted in significant decline in the rhizosphere population of Fusarium oxysporum f . sp . lycopersici . A significant decrease in the severity of wilt occurred with A . awamori (37.1%) and P . digitatum (21.3%) compared to the control . Root-dip treatment with the phosphate solubilizing microorganisms tested resulted in significant increase in the yield of tomato, being greatest with A . awamori and P . digitatum in pathogen inoculated (36% and 33%) and uninoculated plants (19% and 23%) . A chemical fungicide gave 24% better yield.

Can J Microbiol, 2002 Jul, 48(7), 588 - 601
Survival of the rhizosphere-competent biocontrol strain Pseudomonas fluorescens NBRI2650 in the soil and phytosphere; Nautlyal CS et al.; Pseudomonas fluorescens NBRI2650 was isolated after screening 360 bacterial strains from the rhizosphere of chickpea (Cicer arietinum L.) grown in fungal-disease-suppressive field soil . The strain was selected because of its high rhizosphere competence and ability to inhibit the growth of Fusarium oxysporum f.sp . ciceri, Rhizoctonia bataticola, and Pythium sp . under in vitro conditions . Survival and colonization of NBRI2650 in the phytosphere of chickpea, cotton (Gossypium hirsutum L.), cucumber (Cucumis sativus L.), and tomato (Lycopersicon seculentum Mill.) were monitored using a chromosomally located rifampicin-marked mutant P . fluorescens NBRI2650R . The strain showed variable ability to invade and survive in the phytosphere of different plants . Chickpea was used as a tester plant for further work, as it was not invaded by NBRI2650R . The interaction between NBRI2650R and F oxysporum fsp . ciceri was studied by both light microscopy and scanning electron microscopy . The lysis of the fungal cell wall by NBRI2650R was clearly demonstrated . Treatment of the chickpea seeds with NBRI2650R in prerelease experiments in the greenhouse using disease-conducive field soils from Jhansi and Kanpur resulted in increased plant growth and did not result in any perturbation of the indigenous microbial community that inhabited the rhizosphere of chickpea compared with nonbacterized seeds . Direct fermentation of diluted NBRI2650R on vermiculite without the need of expensive fermentors offers a reliable process for manufacturing bacterial inoculants in developing countries . Under field conditions, the horizontal and vertical movement of NBRI2650R was restricted to 30 and 60 cm, respectively, and the strain could not survive in the field during the 7 months before the chickpea could be planted for next cropping season . Field trials conducted at Jhansi, Kanpur, and Pantnagar resulted in higher grain yield increase in the bacteria-treated seed compared with the nonbacterized control . Seed and furrow treatment of the two chickpeas ('Radhey' and 'H-208') at Pantnagar resulted in significantly (P = 0.05) greater seedling mortality in nonbacterized seedlings compared with bacterized ones . The seed dry weight and yield for each variety were also significantly higher in bacterized seedlings than in nonbacterized ones . The population of NBRI2650R persisted throughout the growing season of chickpea in the range of 5.4-6.4 log10 CFU/g root.

J Dairy Res, 2002 May, 69(2), 243 - 54
Manothermosonication of heat-resistant lipase and protease from Pseudomonas fluorescens: effect of pH and sonication parameters; Vercet A et al.; The effect of different parameters (pH, ultrasonic amplitude and pressure) on the resistance to heat and manothermosonication (MTS) treatments of heat resistant lipase and protease produced by Pseudomonas fluorescens B52 and NCDO 2085, respectively, were studied . Lipase B52 thermoresistance decreases with an increase of pH . However, inactivation by MTS seems to be pH independent . There were only slight increases in the MTS efficiency when increasing pressure at UHT temperatures and the effect of amplitude was different depending on treatment temperature . Protease NCDO 2085, which was very resistant to MTS at 30 degrees C . was very sensitive to MTS at 76 degrees C . Increases in applied pressure had no effect on MTS efficiency at 140 degrees C and its inactivation by MTS was almost temperature independent between 76-109 degrees C . Data obtained are compared with previous published data and inactivation mechanisms are discussed.

Gastroenterology, 2002 Sep, 123(3), 689 - 99
Selected loss of tolerance evidenced by Crohn's disease-associated immune responses to auto- and microbial antigens; Landers CJ et al.; BACKGROUND & AIMS: Previous studies in Crohn's disease suggest global loss of tolerance with sonicated bacteria preparations containing hundreds of antigens . Monoassociation studies show that a solitary bacterium can induce colitis in one animal model, whereas another is responsible in other models . Among patients with Crohn's disease, serum responses have been documented to microbial and autoantigens (antibodies to the Escherichia coli outer-membrane porin C and the Pseudomonas fluorescens-associated sequence I2, antisaccharomyces cerevisiae antibody (ASCA), and perinuclear antineutrophil cytoplasmic antibodies) . Our aim was to determine whether there are heterogeneous responses to these specific antigens . METHODS: Sera from 330 Crohn's patients were analyzed . Immunoglobulin A enzyme-linked immunosorbent assays to ASCA, outer-membrane porin C, or I2 and immunoglobulin G enzyme-linked immunosorbent assay to ASCA and ANCA determined the presence and level of antibodies . Perinuclear antineutrophil cytoplasmic antibodies were determined by immunofluorescence . RESULTS: ASCA was detected in 56% of patients; 55% were seroreactive to outer-membrane porin C, 50% were seroreactive to I2, and 23% were perinuclear antineutrophil cytoplasmic antibody positive . Eighty-five percent responded to at least 1 antigen; only 4% responded to all 4 . Among microbial antigens, 78% responded to at least 1, and 57% were double positive, but only 26% responded to all 3 . The level of response was stable over time and with change in disease activity . Among patients with the same qualitative antigen-response profiles, quantitative response differed . Cluster analysis of these antibody responses yielded 4 groups: ASCA, outer-membrane porin C/I2, perinuclear antineutrophil cytoplasmic antibodies, or no/low response . CONCLUSIONS: Rather than global loss of tolerance, there seem to be patient subsets with differing responses to selected microbial and autoantigens.

J Biol Chem, 2002 Nov 8, 277(45), 43433 - 42 Epub 2002 Aug 23.
Crystal structure of Pseudomonas fluorescens mannitol 2-dehydrogenase binary and ternary complexes . Specificity and catalytic mechanism; Kavanagh KL et al.; Long-chain mannitol dehydrogenases are secondary alcohol dehydrogenases that are of wide interest because of their involvement in metabolism and potential applications in agriculture, medicine, and industry . They differ from other alcohol and polyol dehydrogenases because they do not contain a conserved tyrosine and are not dependent on Zn(2+) or other metal cofactors . The structures of the long-chain mannitol 2-dehydrogenase (54 kDa) from Pseudomonas fluorescens in a binary complex with NAD(+) and ternary complex with NAD(+) and d-mannitol have been determined to resolutions of 1.7 and 1.8 A and R-factors of 0.171 and 0.176, respectively . These results show an N-terminal domain that includes a typical Rossmann fold . The C-terminal domain is primarily alpha-helical and mediates mannitol binding . The electron lone pair of Lys-295 is steered by hydrogen-bonding interactions with the amide oxygen of Asn-300 and the main-chain carbonyl oxygen of Val-229 to act as the general base . Asn-191 and Asn-300 are involved in a web of hydrogen bonding, which precisely orients the mannitol O2 proton for abstraction . These residues also aid in stabilizing a negative charge in the intermediate state and in preventing the formation of nonproductive complexes with the substrate . The catalytic lysine may be returned to its unprotonated state using a rectifying proton tunnel driven by Glu-292 oscillating among different environments . Despite low sequence homology, the closest structural neighbors are glycerol-3-phosphate dehydrogenase, N-(1-d-carboxylethyl)-l-norvaline dehydrogenase, UDP-glucose dehydrogenase, and 6-phosphogluconate dehydrogenase, indicating a possible evolutionary relationship among these enzymes.

J Environ Monit, 2002 Aug, 4(4), 482 - 9
Evaluation of interactive toxicity of chlorophenols in water and soil using lux-marked biosensors; Tiensing T et al.; An assessment of the toxicity of three chlorophenol compounds (2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP)), individually and in combinations, was made in deionised water, soil extracts and soils using the lux-marked microbial biosensors: Escherichia coli HB101 pUCD607 and Pseudomonas fluorescens 10586r pUCD607 . These biosensors responded to the bioavailable fraction of pollutants enabling a rapid and ecologically relevant toxicity test . Toxicity interaction responses of pollutant mixtures were predicted after individual compounds were assessed . Synergistic interactions were observed in the response of P . fluorescens to all combinations of chlorophenols tested, while the toxicity response of E . coli varied with the matrices tested . Soil characteristics influenced the toxicity response when compared with aqueous solutions . These results highlight the significance of interactive factors and physicochemical parameters when evaluating toxicity . To develop an understanding of pollution derived hazard assessment in soils we need to integrate a wide spectrum of parameters.

Protein Sci, 2002 Sep, 11(9), 2184 - 95
Crystal structures of a meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with cleavage products; Fushinobu S et al.; 2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase (CumD) from Pseudomonas fluorescens IP01 hydrolyzes a meta-cleavage product generated in the cumene (isopropylbenzene) degradation pathway . The crystal structures of the inactive S103A mutant of the CumD enzyme complexed with isobutyrate and acetate ions were determined at 1.6 and 2.0 A resolution, respectively . The isobutyrate and acetate ions were located at the same position in the active site, and occupied the site for a part of the hydrolysis product with CumD, which has the key determinant group for the substrate specificity of related hydrolases . One of the oxygen atoms of the carboxyl group of the isobutyrate ion was hydrogen bonded with a water molecule and His252 . Another oxygen atom of the carboxyl group was situated in an oxyanion hole formed by the two main-chain N atoms . The isopropyl group of the isobutyric acid was recognized by the side-chains of the hydrophobic residues . The substrate-binding pocket of CumD was long, and the inhibition constants of various organic acids corresponded well to it . In comparison with the structure of BphD from Rhodococcus sp . RHA1, the structural basis for the substrate specificity of related hydrolases, is revealed.

Arch Latinoam Nutr, 2002 Jun, 52(2), 160 - 6
{Purification and characterization of proteases from Pseudomonas fluorescens and their effects on milk proteins}; Costa M et al.; The increase in the levels of psychotropic bacteria in the raw milk during the refrigeration period, could lead to the production of heat-resistant enzymes responsible for the deterioration of long-life industrial dairy products . Pseudomonas fluorescens is the psychotropic bacteria most commonly found in milk in Southern Chile . In the present work the enzymatic proteinases extract of cultures of Pseudomonas fluorescens RV10 at 6 degrees C in raw milk just milked were purified . It was found that the proteases corresponds to a protein with a molecular mass of 49.5 kD, that presents heat resistance and rapidly attacks the k-casein continuing with the b-casein . It is possible to conclude that storage of the milk for long-life products at 6 degrees C is risky, as it causes the loss of quality for the proteases of psychotropic bacteria.

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