Alginate-perlite encapsulated Pseudomonas putida A (ATCC 12633) cells: Preparation, characterization and potential use as plant inoculants.

Microbial immobilization can be used to prepare encapsulated inoculants. Here, we characterize and describe the preparation of Ca-alginate-perlite microbeads loaded with cells of plant growth-promoting Pseudomonas putida A (ATCC 12633), for their future application as agricultural inoculants. The microbeads were prepared by dropwise addition of a CaCl2-paraffin emulsion mixture to an emulsion containing alginate 2% (w/v), perlite 0.1-0.4% (w/v) and bacterial suspension in 0.9% NaCl (1010 CFU/mL). For all perlite concentrations used, microbead size was 90-120 µm, the trapped population was 108 CFU/g microbeads and the increase in mechanical stability was proportional to perlite concentration. Microbeads containing 0.4% (w/v) perlite were able to release bacteria into the medium after 30 days of incubation. When we evaluated how P. putida A (ATCC 12633) entrapped in Ca-alginate-perlite (0.4% (w/v)) microbeads colonized the Arabidopsis thaliana rhizosphere, an increase in colonization over time was detected (from an initial 2.1 × 104 to 9.2 × 105 CFU/g soil after 21 days). With this treatment, growth promotion of A. thaliana occurred with an increase in the amount of proteins, and in root and leaf biomass. It was concluded that the microbeads could be applied as possible inoculants, since they provide protection and a controlled release of microorganisms into the rhizosphere.

Biofilm as a production platform for heterologous production of rhamnolipids by the non-pathogenic strain Pseudomonas putida KT2440.

BACKGROUND: Although a transition toward sustainable production of chemicals is needed, the physiochemical properties of certain biochemicals such as biosurfactants make them challenging to produce in conventional bioreactor systems. Alternative production platforms such as surface-attached biofilm populations could potentially overcome these challenges. Rhamnolipids are a group of biosurfactants highly relevant for industrial applications. However, they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa using hydrophobic substrates such as plant oils. As the biosynthesis is tightly regulated in P. aeruginosa a heterologous production of rhamnolipids in a safe organism can relive the production from many of these limitations and alternative substrates could be used. RESULTS: In the present study, heterologous production of biosurfactants was investigated using rhamnolipids as the model compound in biofilm encased Pseudomonas putida KT2440. The rhlAB operon from P. aeruginosa was introduced into P. putida to produce mono-rhamnolipids. A synthetic promoter library was used in order to bypass the normal regulation of rhamnolipid synthesis and to provide varying expression levels of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology. Quantitative and structural investigations of the produced rhamnolipids were made by ultra performance liquid chromatography combined with high resolution mass spectrometry (HRMS) and tandem HRMS. The predominant rhamnolipid congener produced by the heterologous P. putida biofilm was mono-rhamnolipid with two C10 fatty acids. CONCLUSION: This study shows a successful application of synthetic promoter library in P. putida KT2440 and a heterologous biosynthesis of rhamnolipids in biofilm encased cells without hampering biofilm capabilities. These findings expands the possibilities of cultivation setups and paves the way for employing biofilm flow systems as production platforms for biochemicals, which as a consequence of physiochemical properties are troublesome to produce in conventional fermenter setups, or for production of compounds which are inhibitory or toxic to the production organisms.

Interactive effects of phosphorus and Pseudomonas putida on chickpea (Cicer arietinum L.) growth, nutrient uptake, antioxidant enzymes and organic acids exudation.

Phosphorus (P) availability in alkaline soils of arid and semi-arid regions is a major constraint for decreased crop productivity. Use of plant growth promoting rhizobacteria (PGPR) may enhance plant growth through the increased plant antioxidation activity. Additionally, PGPR may increase nutrient uptake by plants as a result of induced root exudation and rhizosphere acidification. The current study was aimed to investigate combined effects of P and Pesudomonas putida (PGPR) on chickpea growth with reference to antioxidative enzymatic activity and root exudation mediated plant nutrient uptake, particularly P. Half of the seeds were soaked in PGPR solution, whereas others in sterile water and latter sown in soils. Plants were harvested 8 weeks after onset of experiment and analyzed for leaf nutrient contents, antioxidant enzymes activities and organic acids concentrations. Without PGPR, P application (+P) increased various plant growth attributes, plant uptake of P and Ca, soil pH, citric acid and oxalic acid concentrations, whereas decreased the leaf POD enzymatic activity as compared to the P-deficiency. PGPR supply both under -P and +P improved the plant growth, plant uptake of N, P, and K, antioxidative activity of SOD and POD enzymes and concentrations of organic acids, whereas reduced the rhizosphere soil pH. Growth enhancement by PGPR supply was related to higher plant antioxidation activity as well as nutrient uptake of chickpea including P as a result of root exudation mediated rhizosphere acidification.

Biocontrol of the Potato Blackleg and Soft Rot Diseases Caused by Dickeya dianthicola.

Development of protection tools targeting Dickeya species is an important issue in the potato production. Here, we present the identification and the characterization of novel biocontrol agents. Successive screenings of 10,000 bacterial isolates led us to retain 58 strains that exhibited growth inhibition properties against several Dickeya sp. and/or Pectobacterium sp. pathogens. Most of them belonged to the Pseudomonas and Bacillus genera. In vitro assays revealed a fitness decrease of the tested Dickeya sp. and Pectobacterium sp. pathogens in the presence of the biocontrol agents. In addition, four independent greenhouse assays performed to evaluate the biocontrol bacteria effect on potato plants artificially contaminated with Dickeya dianthicola revealed that a mix of three biocontrol agents, namely, Pseudomonas putida PA14H7 and Pseudomonas fluorescens PA3G8 and PA4C2, repeatedly decreased the severity of blackleg symptoms as well as the transmission of D. dianthicola to the tuber progeny. This work highlights the use of a combination of biocontrol strains as a potential strategy to limit the soft rot and blackleg diseases caused by D. dianthicola on potato plants and tubers.

Impact of dissolved organic matter on bacterial tactic motility, attachment, and transport.

Bacterial dispersal is a key driver of the ecology of microbial contaminant degradation in soils. This work investigated the role of dissolved organic matter (DOM) in the motility, attachment, and transport of the soil bacterium Pseudomonas putida G7 in saturated porous media. The study is based on the hypothesis that DOM quality is critical to triggering tactic motility and, consequently, affects bacterial transport and dispersal. Sunflower root exudates, humic acids (HA), and the synthetic oleophilic fertilizer S-200 were used as representatives of fresh, weathered, and artificially processed DOM with high nitrogen and phosphorus contents, respectively. We studied DOM levels of 16-130 mg L(-1), which are representative of DOM concentrations typically found in agricultural soil pore water. In contrast to its responses to HA and S-200, strain G7 exhibited a tactic behavior toward root exudates, as quantified by chemotaxis assays and single-cell motility observations. All DOM types promoted bacterial transport through sand at high concentrations (∼ 130 mg L(-1)). At low DOM concentrations (∼ 16 mg L(-1)), the enhancement occurred only in the presence of sunflower root exudates, and this enhancement did not occur with G7 bacteria devoid of flagella. Our results suggest that tactic DOM effectors strongly influence bacterial transport and the interception probability of motile bacteria by collector surfaces.

Nutrition effects on the biofilm immobilization and 3,5-DNBA degradation of Comamonas testosteroni A3 during bioaugmentation treatment.

The survival of inoculated microbes is critical for successful bioaugmentation in wastewater treatment. The influence of readily available nutrients (RANs) on the colonization of two functional bacteria, Pseudomonas putida M9, a strong biofilm-forming strain, and Comamonas testosteroni A3, a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading strain, in biofilms was studied with 3,5-dinitrobenoic acid synthetic wastewater (DCMM) complemented with various ratios of Luria-Bertani broth (LB). With the increase in LB rate, the biofilm biomass was increased, the percentage of gfp-labeled M9 measured in the mixed culture enhanced, and also M9 became dominant. In laboratory-scale sequencing batch biofilm reactors, with the increase in 3,5-DNBA concentration and extension of the running time, the 3,5-DNBA removal in DCMM wastewater complemented with RANs tended to be more efficient and its removal rates increased gradually over the experimental period. Our study demonstrated that supplementing RANs could be a useful strategy for enhancing colonization of degrading bacteria in wastewater treatment systems.

ATR-FTIR spectroscopic evidence for biomolecular phosphorus and carboxyl groups facilitating bacterial adhesion to iron oxides.

Attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy has been used to probe the binding of bacteria to hematite (α-Fe2O3) and goethite (α-FeOOH). In situ ATR-FTIR experiments with bacteria (Pseudomonas putida, Pseudomonas aeruginosa, Escherichia coli), mixed amino acids, polypeptide extracts, deoxyribonucleic acid (DNA), and a suite of model compounds were conducted. These compounds represent carboxyl, catecholate, amide, and phosphate groups present in siderophores, amino acids, polysaccharides, phospholipids, and DNA. Due in part to the ubiquitous presence of carboxyl groups in biomolecules, numerous IR peaks corresponding to outer-sphere or unbound (1400 cm(-1)) and inner-sphere (1310-1320 cm(-1)) coordinated carboxyl groups are noted following reaction of bacteria and biomolecules with α-Fe2O3 and α-FeOOH. However, the data also reveal that the presence of low-level amounts (i.e., 0.45-0.79%) of biomolecular phosphorous groups result in strong IR bands at ∼1043 cm(-1), corresponding to inner-sphere Fe-O-P bonds, underscoring the importance of bacteria associated P-containing groups in biomolecule and cell adhesion. Spectral comparisons also reveal slightly greater P-O-Fe contributions for bacteria (Pseudomonad, E. coli) deposited on α-FeOOH, as compared to α-Fe2O3. This data demonstrates that slight differences in bacterial adhesion to Fe oxides can be attributed to bacterial species and Fe-oxide minerals. However, more importantly, the strong binding affinity of phosphate in all bacteria samples to both Fe-oxides results in the formation of inner-sphere Fe-O-P bonds, signifying the critical role of biomolecular P in the initiation of bacterial adhesion.

Pb remobilization by bacterially mediated dissolution of pyromorphite Pb5(PO4)3Cl in presence of phosphate-solubilizing Pseudomonas putida.

Remediation of lead (Pb)-contaminated sites with phosphate amendments is one of the best studied and cost-effective methods for in situ immobilization. In this treatment, a very stable mineral, pyromorphite Pb5(PO4)3Cl, is formed. Several studies propose to improve this treatment method with the addition of phosphate-solubilizing bacteria (PSB). The effect of bacteria on solubilization of pyromorphite is unknown. In this study, the effect of the soil microorganisms on the stability of pyromorphite Pb5(PO4)3Cl has been investigated in a set of batch solution experiments. The mineral was reacted with Pseudomonas putida, a common soil microorganism. Dissolution of pyromorphite was enhanced by the presence of P. putida, resulting in an elevated Pb concentration in the solution. This occurred even when the bacteria were provided with an additional source of phosphate in the solution. Pyromorphite has been shown to be a potential source of nutrient phosphorus for common soil bacteria. Thus, the use of PSB in remediation treatments of Pb contaminated sites may have adverse long-term impacts on Pb immobilization. Conscious phosphate management is suggested for long-term sustainability of the in situ Pb immobilization by pyromorphite formation.

Investigating Pseudomonas putida-Candida humicola interactions as affected by chelate Fe(III) in soil.

Microcalorimetric technique was applied to assess the toxic effect of EDTA-chelated trivalent iron on Pseudomonas putida (P. putida) (bacterium), Candida humicola (C. humicola) (fungus) and their mixture in sterilized soil. Microbial growth rate constant k, total heat evolution Q T, metabolic enthalpy ∆H met, mass specific heat rate J Q/S, microbial biomass C and inhibitory ratio I were calculated. Results showed that microcalorimetric indexes decreased with the increasing Fe(III)-EDTA complex concentration. Comparing the single and mixed strains, the effect of Fe(III) on bacterium-fungus interaction was dominant at lower dose, whereas, the metal toxicity at high dose of Fe was the main factor affecting P. putida and C. humicola activity. Thus, the mixture had moderate tolerance to the iron overload, and exhibit synergistic interaction in exponential growth phase (0-0.3 mg g(-1)). The results of glucose degradation showed that glucose was consumed totally at the end of exponential phase of microbial growth.

Tactic responses to pollutants and their potential to increase biodegradation efficiency.

A significant number of bacterial strains are able to use toxic aromatic hydrocarbons as carbon and energy sources. In a number of cases, the evolution of the corresponding degradation pathway was accompanied by the evolution of tactic behaviours either towards or away from these toxic carbon sources. Reports are reviewed which show that a chemoattraction to heterogeneously distributed aromatic pollutants increases the bioavailability of these compounds and their biodegradation efficiency. An extreme form of chemoattraction towards aromatic pollutants, termed 'hyperchemotaxis', was described for Pseudomonas putida DOT-T1E, which is based on the action of the plasmid-encoded McpT chemoreceptor. Cells with this phenotype were found of being able to approach and of establishing contact with undiluted crude oil samples. Although close McpT homologues are found on other degradation plasmids, the sequence of their ligand-binding domains does not share significant similarity with that of NahY, the other characterized chemoreceptor for aromatic hydrocarbons. This may suggest the existence of at least two families of chemoreceptors for aromatic pollutants. The use of receptor chimers comprising the ligand-binding region of McpT for biosensing purposes is discussed.

Are leaf glandular trichomes of oregano hospitable habitats for bacterial growth?

Phyllospheric bacteria were isolated from microsites around essential-oil-containing glands of two oregano (Origanum vulgare subsp. hirtum) lines. These bacteria, 20 isolates in total, were subjected to bioassays to examine their growth potential in the presence of essential oils at different concentrations. Although there were qualitative and quantitative differences in the essential oil composition between the two oregano lines, no differences were recorded in their antibacterial activity. In disk diffusion bioassays, four of the isolated strains could grow almost unrestrained in the presence of oregano oil, another five proved very sensitive, and the remaining 11 showed intermediate sensitivity. The strain least inhibited by oregano essential oil was further identified by complete16s rRNA gene sequencing as Pseudomonas putida. It was capable of forming biofilms even in the presence of oregano oil at high concentrations. Resistance of P. putida to oregano oil was further elaborated by microwell dilution bioassays, and its topology on oregano leaves was studied by electron microscopy. When inoculated on intact oregano plants, P. putida was able not only to colonize sites adjacent to essential oil-containing glands, but even to grow intracellularly. This is the first time that such prolific bacterial growth inside the glands has been visually observed. Results of this study further revealed that several bacteria can be established on oregano leaves, suggesting that these bacteria have attributes that allow them to tolerate or benefit from oregano secondary metabolites.

Simultaneous removal of phosphorus and nitrogen in a sequencing batch biofilm reactor with transgenic bacteria expressing polyphosphate kinase.

To improve phosphorus removal from wastewater, we constructed a high-phosphate-accumulating microorganism, KTPPK, using Pseudomonas putida KT2440 as a host. The expression plasmid was constructed by inserting and expressing polyphosphate kinase gene (ppk) from Microcystis aeruginosa NIES-843 into broad-host-range plasmid, pBBR1MCS-2. KTPPK was then added to a sequencing batch biofilm reactor (SBBFR) using lava as a biological carrier. The results showed that SBBFR with KTPPK not only efficiently removed COD, NH(3)-N, and NO(3)(-)-N but also had a high removal capacity for PO(4)(3-)-P, resulting in a low phosphorus concentration remaining in the outflow of the SBBFR. The biofilm increased by 30-53% on the lava in the SBBFR that contained KTPPK after 11 days when compared with the reactor that contained P. putida KT2440. Real-time quantitative polymerase chain reaction confirmed that the copy of ppk was maintained at about 3.5 × 10(10) copies per µL general DNA in the biofilm after 20 days. Thus, the transgenic bacteria KTPPK could maintain a high density and promote phosphorus removal in the SBBFR. In summary, this study indicates that the use of SBBFR with transgenic bacteria has the potential to remove phosphorus and nitrogen from wastewater.

Suppressing Erwinia carotovora pathogenicity by projecting N-acyl homoserine lactonase onto the surface of Pseudomonas putida cells.

N-Acyl homoserine lactones (AHLs) serve as the vital quorum-sensing signals that regulate the virulence of the pathogenic bacterium Erwinia carotovora. In the present study, an approach to efficiently restrain the pathogenicity of E. carotovora-induced soft rot disease is described. Bacillus thuringiensis-derived N-acyl homoserine lactonase (AiiA) was projected onto the surface of Pseudomonas putida cells, and inoculation with both strains was challenged. The previously identified N-terminal moiety of the ice nucleation protein, InaQ-N, was applied as the anchoring motif. A surface display cassette with inaQ-N/ aiiA was constructed and expressed under the control of a constitutive promoter in P. putida AB92019. Surface localization of the fusion protein was confirmed by Western blot analysis, flow cytometry, and immunofluorescence microscopy. The antagonistic activity of P. putida MB116 expressing InaQ-N/AiiA toward E. carotovora ATCC25270 was evaluated by challenge inoculation in potato slices at different ratios. The results revealed a remarkable suppressing effect on E. carotovora infection. The active component was further analyzed using different cell fractions, and the cell surface-projected fusion protein was found to correspond to the suppressing effect.

Endophytic colonization of potato (Solanum tuberosum L.) by a novel competent bacterial endophyte, Pseudomonas putida strain P9, and its effect on associated bacterial communities.

Pseudomonas putida strain P9 is a novel competent endophyte from potato. P9 causes cultivar-dependent suppression of Phytophthora infestans. Colonization of the rhizoplane and endosphere of potato plants by P9 and its rifampin-resistant derivative P9R was studied. The purposes of this work were to follow the fate of P9 inside growing potato plants and to establish its effect on associated microbial communities. The effects of P9 and P9R inoculation were studied in two separate experiments. The roots of transplants of three different cultivars of potato were dipped in suspensions of P9 or P9R cells, and the plants were planted in soil. The fate of both strains was followed by examining colony growth and by performing PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Colonies of both strains were recovered from rhizoplane and endosphere samples of all three cultivars at two growth stages. A conspicuous band, representing P9 and P9R, was found in all Pseudomonas PCR-DGGE fingerprints for treated plants. The numbers of P9R CFU and the P9R-specific band intensities for the different replicate samples were positively correlated, as determined by linear regression analysis. The effects of plant growth stage, genotype, and the presence of P9R on associated microbial communities were examined by multivariate and unweighted-pair group method with arithmetic mean cluster analyses of PCR-DGGE fingerprints. The presence of strain P9R had an effect on bacterial groups identified as Pseudomonas azotoformans, Pseudomonas veronii, and Pseudomonas syringae. In conclusion, strain P9 is an avid colonizer of potato plants, competing with microbial populations indigenous to the potato phytosphere. Bacterization with a biocontrol agent has an important and previously unexplored effect on plant-associated communities.

Effect of combined nutrients on biosurfactant produced by Pseudomonas putida.

This work investigated biosurfactant production by Pseudomonas putida in combined C/P, C/Ninorganic, C/Fe, C/Mg nutrient ratios and peptone concentration. Analysis of the 2(5-1) fractional factorial experimental design showed that only the C/Fe ratio had a significant (p<0.02) effect on biosurfactant production. The highest amount of biosurfactant was obtained at low C/Fe ratios, but net surface tension did not show significant differences. In addition, low amounts of peptone and the C/P-C/Mg nutrient ratios interaction significantly (p < 0.05) enhanced the biomass produced by P. putida. Analysis of biosurfactant by gas chromatography (GC) showed that the hydrophilic fraction was composed by rhamnose and the hydrophobic fraction, mainly by palmitic (C16), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) fatty acids.

Comparison of application methods to prolong the survival of potential biocontrol bacteria on stored sugar-beet seed.

AIMS: To develop bacterial inoculation treatments on sugar-beet seed that will maintain a commercially acceptable degree of viability for a minimum of 4 months storage at ambient temperature. METHODS AND RESULTS: Single rifampicin-resistant (Rif(+)) strains of both Gram-positive and negative bacterial isolates (mostly pseudomonads) were applied in turn to sugar-beet seed in a comparative study by seed soaking, encapsulation in alginate, pelleting using an inoculated peat carrier or seed priming. The treated seed was assessed for bacterial survival over a time course by plating out homogenized samples onto a selective medium. Priming inoculation offered a significant improvement over all the other application strategies tested. After pelleting with fungicides and drying at 40 degrees C, Pseudomonas marginalis/putida P1W1 maintained populations of >6.6 log(10) CFU g(-1) seed during 4 months storage at 15 degrees C. Subsequent experiments verified a stabilized population under these storage conditions with commercial pellets at <7% moisture content. CONCLUSION: An inoculation method was established which allowed the survival on seed of a Gram-negative bacterium at ambient temperature with little loss in viability. SIGNIFICANCE AND IMPACT OF THE STUDY: This has promising implications for the delivery of beneficial bacteria, especially Gram-negative strains, on sugar beet.

Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants.

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards VERTICILLIUM: The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.

Dual system to reinforce biological containment of recombinant bacteria designed for rhizoremediation.

Active biological containment (ABC) systems have been designed to control at will the survival or death of a bacterial population. These systems are based on the use of a killing gene, e.g., a porin-inducing protein such as the one encoded by the Escherichia coli gef gene, and a regulatory circuit that controls expression of the killing gene in response to the presence or absence of environmental signals. An ABC system for recombinant microorganisms that degrade a model pollutant was designed on the basis of the Pseudomonas putida TOL plasmid meta-cleavage regulatory circuit. The system consists of a fusion of the Pm promoter to lacI, whose expression is controlled by XylS with 3-methylbenzoate, and a fusion of a synthetic P(lac) promoter to gef. In the presence of the model pollutant, bacterial cells survived and degraded the target compound, whereas in the absence of the aromatic carboxylic acid cell death was induced. The system had two main drawbacks: (i) the slow death of the bacterial cells in soil versus the fast killing rate in liquid cultures in laboratory assays, and (ii) the appearance of mutants, at a rate of about 10(-8) per cell and generation, that did not die after the pollutant had been exhausted. We reinforced the ABC system by including it in a Deltaasd P. putida background. A P. putida Deltaasd mutant is viable only in complex medium supplemented with diaminopimelic acid, methionine, lysine, and threonine. We constructed a P. putida Deltaasd strain, called MCR7, with a Pm::asd fusion in the host chromosome. This strain was viable in the presence of 3-methylbenzoate because synthesis of the essential metabolites was achieved through XylS-dependent induction. In the P. putida MCR7 strain, an ABC system (Pm::lacI, xylS, P(lac)::gef) was incorporated into the host chromosome to yield strain MCR8. The number of MCR8 mutants that escaped killing was below our detection limit (<10(-9) mutants per cell and generation). The MCR8 strain survived and colonized rhizosphere soil with 3-methylbenzoate at a level similar to that of the wild-type strain. However, it disappeared in less than 20 to 25 days in soils without the pollutant, whereas an asd(+), biologically contained counterpart such as P. putida CMC4 was still detectable in soils after 100 days.

Inorganic nutrient utilisation by "adapted" Pseudomonas putida strain used in the bioremediation of agricultural soil polluted with crude petroleum.

Garden soil samples polluted with crude petroleum were bioremediated by inorganic nutrient monitoring with appropriate adjustment and inoculation with crude oil-adapted strain of Pseudomonasputida (PP) isolated from oil-impacted soils. Soil samples without PP inoculation served as the control samples to compare the abilities of the native soil microflora with the adapted PP strain in biodegrading crude oil pollutant. In the experimental samples, oil concentration and all the inorganic nutrient sources tested decreased more rapidly with a proportional increase in the population densities of both PP and the native soil microflora than were observed in the control samples. This trend was particularly strong for PO4(3-) and NO3- which eventually became limiting both in all the experimental samples and in some control samples. Inoculation of crude oil-impacted agricultural soils by oil -adapted PP strain with nutrient monitoring and adjustment can be effective as bioremediation methods of agricultural land upon pollution with petroleum or petroleum products.

Expression of the aggA locus of Pseudomonas putida in vitro and in planta as detected by the reporter gene, xylE.

In vitro agglutinability by Pseudomonas putida, isolate Corvallis, with a plant root surface agglutinin is correlated with rapid adhesion of cells of the fluorescent pseudomonad to bean (Phaseolus vulgaris) root surfaces. Agglutinability in P. putida cells is regulated by nutrient status as well as growth phase. Cells grown in three different nutrient complex media are agglutinable at early and mid-late logarithmic phase but become nonagglutinable at stationary phase. Cells grown in a minimal medium are weakly agglutinable, but the addition of lysine, aspartic acid, or histidine increases agglutinability. Cells in the same minimal medium supplemented with bean root surface components grow in a highly agglutinated state. Previous data indicate both agglutination and rapid adhesion to roots by P. putida Corvallis involves the aggA locus, which contains two putative open reading frames (ORF), ORF-AGG1 and ORFAGG2, on complementary strands. Sequence and deletion analyses suggest ORFAGG1 is the most probable ORF responsible for agglutination and adhesion. Chimeric fusion of an Escherichia coli lac promoter with ORFAGG1, but not with ORFAGG2, complemented agglutinability of an aggA::Tn5 P. putida Agg mutant, providing further evidence that ORFAGG1, not ORFAGG2, is responsible for agglutination. Heterologous expression of ORFAGG1 yields a 50-kDa precursor and a 48-kDa mature periplasmic protein. Fusions of ORFAGG1 and ORFAGG2 to the reporter gene, xylE, and detection of the reporter enzyme, catechol-2,3-oxygenase reveal an active promoter in the 5' noncoding region of ORFAGG1. The ORFAGG1 promoter is active during growth of the cells in liquid culture and is regulated by growth medium. Greatest activity of the catechol-2,3-oxygenase is observed in stationary phase when the cells are nonagglutinable. Expression of the ORFAGG1 promoter is detected in P. putida cells extracted from the root surface of bean at 48 and 72 hr after inoculation.