Showing papers on "Pseudomonas putida published in 2007"
TL;DR: With the advancement in technology for enzyme immobilization, it is speculated that the direct application of Cr6+ reductases may be a promising approach for bioremediation ofCr6+ in a wide range of environments.
588 citations
TL;DR: The reduction in the detrimental effect of IAA on root elongation could be associated with a reduced ethylene production resulting from a decrease of its precursor 1-aminocyclopropane-1-carboxylic acid by microbial degradation in the rhizosphere and/or by ACC deaminase activity present in both microorganisms.
Abstract: Five bacteria (Pseudomonas fluorescens, P. fluorescens subgroup G strain 2, P. marginalis, P. putida subgroup B strain 1 and P. syringae strain 1) and three fungi (Penicillium brevicompactum, P. solitum strain 1 and Trichoderma atroviride) were evaluated to determine their promoting effect on the growth of mature healthy tomato plants grown under hydroponic conditions. P. putida and T. atroviride were shown to improve fruit yields in rockwool and in organic medium. The production or degradation of indole acetic acid (IAA) by the two microorganisms was investigated as possible mechanisms for plant growth stimulation. Both P. putida and T. atroviride were shown to produce IAA. The production of IAA by the two microorganisms was stimulated in vitro by the addition of l -tryptophan, tryptamine and tryptophol (200 μg ml−1) in the culture medium. P. putida and T. atroviride also increased the fresh weight of both the shoot and the roots of tomato seedlings grown in the presence of increasing concentrations of l -tryptophan (up to 0.75 mM). Both microorganisms showed partial degradation of IAA in vitro when grown in a minimal medium with or without sucrose. In addition, the capacity of these microorganisms to reduce the deleterious effect of exogenous IAA was investigated using tomato seedlings. The results showed that the roots of tomato seedlings grown in the presence of increasing concentrations of IAA (0–10 μg ml−1) were significantly longer when seeds were previously treated with P. putida or T. atroviride. The reduction in the detrimental effect of IAA on root elongation could be associated with a reduced ethylene production resulting from a decrease of its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) by microbial degradation of IAA in the rhizosphere and/or by ACC deaminase activity present in both microorganisms.
565 citations
TL;DR: The findings suggest that in response to water-limiting conditions, pseudomonads produce alginate, which influences biofilm development and EPS physiochemical properties, and collectively these responses may facilitate the maintenance of a hydrated microenvironment, protecting residents from desiccation stress and increasing survival.
Abstract: Biofilms exist in a variety of habitats that are routinely or periodically not saturated with water, and residents must integrate cues on water abundance (matric stress) or osmolarity (solute stress) into lifestyle strategies. Here we examine this hypothesis by assessing the extent to which alginate production by Pseudomonas putida strain mt-2 and by other fluorescent pseudomonads occurs in response to water limitations and how the presence of alginate in turn influences biofilm development and stress tolerance. Total exopolysaccharide (EPS) and alginate production increased with increasing matric, but not solute, stress severity, and alginate was a significant component, but not the major component, of EPS. Alginate influenced biofilm architecture, resulting in biofilms that were taller, covered less surface area, and had a thicker EPS layer at the air interface than those formed by an mt-2 algD mutant under water-limiting conditions, properties that could contribute to less evaporative water loss. We examined this possibility and show that alginate reduces the extent of water loss from biofilm residents by using a biosensor to quantify the water potential of individual cells and by measuring the extent of dehydration-mediated changes in fatty acid composition following a matric or solute stress shock. Alginate deficiency decreased survival of desiccation not only by P. putida but also by Pseudomonas aeruginosa PAO1 and Pseudomonas syringae pv. syringae B728a. Our findings suggest that in response to water-limiting conditions, pseudomonads produce alginate, which influences biofilm development and EPS physiochemical properties. Collectively these responses may facilitate the maintenance of a hydrated microenvironment, protecting residents from desiccation stress and increasing survival.
365 citations
TL;DR: The growth of canola plants treated with either wild-type Pseudomonas putida UW4 or a 1-aminocyclopropane-1-carboxylate (ACC) deaminase minus mutant of this strain was monitored in the presence of inhibitory levels of salt, confirming the previous suggestion that bacterial strains that contain ACC deaminases confer salt tolerance to plants by lowering salt-induced ethylene synthesis.
Abstract: The growth of canola plants treated with either wild-type Pseudomonas putida UW4 or a 1-aminocyclopropane-1-carboxylate (ACC) deaminase minus mutant of this strain was monitored in the presence of ...
345 citations
TL;DR: The glucokinase pathway is a sine qua non condition for P. putida to grow with glucose, and the 2-ketogluconate loop were quantitatively more important than the direct phosphorylation of gluconate.
Abstract: In this study, we show that glucose catabolism in Pseudomonas putida occurs through the simultaneous operation of three pathways that converge at the level of 6-phosphogluconate, which is metabolized by the Edd and Eda Entner/Doudoroff enzymes to central metabolites. When glucose enters the periplasmic space through specific OprB porins, it can either be internalized into the cytoplasm or be oxidized to gluconate. Glucose is transported to the cytoplasm in a process mediated by an ABC uptake system encoded by open reading frames PP1015 to PP1018 and is then phosphorylated by glucokinase (encoded by the glk gene) and converted by glucose-6-phosphate dehydrogenase (encoded by the zwf genes) to 6-phosphogluconate. Gluconate in the periplasm can be transported into the cytoplasm and subsequently phosphorylated by gluconokinase to 6-phosphogluconate or oxidized to 2-ketogluconate, which is transported to the cytoplasm, and subsequently phosphorylated and reduced to 6-phosphogluconate. In the wild-type strain, glucose was consumed at a rate of around 6 mmol g(-1) h(-1), which allowed a growth rate of 0.58 h(-1) and a biomass yield of 0.44 g/g carbon used. Flux analysis of (13)C-labeled glucose revealed that, in the Krebs cycle, most of the oxalacetate fraction was produced by the pyruvate shunt rather than by the direct oxidation of malate by malate dehydrogenase. Enzymatic and microarray assays revealed that the enzymes, regulators, and transport systems of the three peripheral glucose pathways were induced in response to glucose in the outer medium. We generated a series of isogenic mutants in one or more of the steps of all three pathways and found that, although all three functioned simultaneously, the glucokinase pathway and the 2-ketogluconate loop were quantitatively more important than the direct phosphorylation of gluconate. In physical terms, glucose catabolism genes were organized in a series of clusters scattered along the chromosome. Within each of the clusters, genes encoding porins, transporters, enzymes, and regulators formed operons, suggesting that genes in each cluster coevolved. The glk gene encoding glucokinase was located in an operon with the edd gene, whereas the zwf-1 gene, encoding glucose-6-phosphate dehydrogenase, formed an operon with the eda gene. Therefore, the enzymes of the glucokinase pathway and those of the Entner-Doudoroff pathway are physically linked and induced simultaneously. It can therefore be concluded that the glucokinase pathway is a sine qua non condition for P. putida to grow with glucose.
241 citations
Journal Article•
TL;DR: The data suggest that seed inoculation of barley with plant PGPR increased root weight by 17.9%-32.1% as compared to the control, and increased shoot weight by 28.8%-54.2%, depending on the species.
Abstract: This study was conducted with barley in greenhouse conditions in order to investigate seed inoculation with 5 different N2-fixing (Bacillus licheniformis RC02, Rhodobacter capsulatus RC04, Paenibacillus polymyxa RC05, Pseudomonas putida RC06, and Bacillus OSU-142) and 2 different phosphate-solubilising (Bacillus megaterium RC01 and Bacillus M-13) bacteria in comparison to control and mineral fertiliser (N and P) application. Among the strains used in the present study, 6 plant growth promoting rhizobacteria (PGPR) stimulated indole acetic acid (IAA) production and 3 of them stimulated phosphate solubilisation; all bacterial strains fixed N2 and significantly increased the growth of barley. Available phosphate in soil was significantly increased by seed inoculation with Bacillus M-13 and B. megaterium RC01. Maximum NO3-N was found in soil after inoculation with N2-fixing Bacillus OSU-142, followed by P. polymyxa RC05 and R. capsulatus RC04. Total culturable bacteria count increased in all treatments with time, whereas N2-fixing bacteria decreased with time, except with B. megaterium RC01 inoculation. The data suggest that seed inoculation of barley with plant PGPR increased root weight by 17.9%-32.1% as compared to the control, and increased shoot weight by 28.8%-54.2%, depending on the species. N2-fixing bacterial inoculation significantly increased uptake of N, Fe, Mn, and Zn by barley. The production of hormones is suggested to be one of the mechanisms by which PGPR stimulate barley growth. Effective Bacillus species, such as OSU-142, RC07, M-13, P. polymyxa RC05, P. putida RC06, and R. capsulatus RC04, may be used in agriculture.
187 citations
TL;DR: The results suggest that electrostatic interactions play a vital role in P. putida adsorption by soil colloidal factions and are of fundamental significance for the understanding of the survival and transport of bacteria in soil systems.
178 citations
TL;DR: The data suggest that the specific stimulation of indigenous fungi is a promising method to mobilize pollutant degrading bacteria and thereby improve soil bioremediation in-situ.
Abstract: The effect of fungal hyphae on the mobilization of soil-dwelling bacteria and their access to hydrophobic phenanthrene in soil was tested in columns containing air-filled agricultural soil. The experimental design included a spatial separation between zones of bacterial inoculation and contamination. Motile Pseudomonas putida PpG7 (NAH7) and fast-growing, hydrophilic Pythium ultimum were used as the model phenanthrene-degrading and vector organisms, respectively. Efficient translocation of strain PpG7 in the range of centimetres in presence of P. ultimum indicated that the fungal mycelia bridged air-filled pores and thereby provided a continuous network of water-paths that enabled bacteria to spread in the soil. Biodegradation of the soil-associated phenanthrene was found only in the presence of the fungal mycelia, hence proving that the fungal network facilitated the access of the bacteria to the contaminant. Our data suggest that the specific stimulation of indigenous fungi is a promising method to mobilize pollutant degrading bacteria and thereby improve soil bioremediation in-situ.
164 citations
TL;DR: Thermal and mechanical properties assays indicated that this new type PHA with a high HDD fraction had higher crystallinity and tensile strength than P HA with a low HDD fraction did, demonstrating an improved application property.
155 citations
TL;DR: This is the first field study to document the increase in plant tolerance utilizing transgenic plants and plant growth-promoting bacteria exposed to multiple stressors.
137 citations
TL;DR: It was concluded that carbon-limited exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA.
Abstract: Pseudomonas putida KT2440 grew on glucose at a specific rate of 0.48 h−1 but accumulated almost no poly-3-hydroxyalkanoates (PHA). Subsequent nitrogen limitation on nonanoic acid resulted in the accumulation of only 27% medium-chain-length PHA (MCL-PHA). In contrast, exponential nonanoic acid-limited growth (μ = 0.15 h−1) produced 70 g l−1 biomass containing 75% PHA. At a higher exponential feed rate (μ = 0.25 h−1), the overall productivity was increased but less biomass (56 g l−1) was produced due to higher oxygen demand, and the biomass contained less PHA (67%). It was concluded that carbon-limited exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA. Nitrogen limitation is unnecessary.
TL;DR: The results suggest that P. putida mt-2 degrades the studied azo dyes in two steps: an azo-reduction followed by an oxygen-dependent metabolization, which would be responsible of genotoxicity and metabolic toxicity.
TL;DR: Investigation showed that contaminated water outlets represented the possible source of spread and two modes of catheter contamination, and a reduction in the size of the catheter dressing at the exit site gave less protective cover during showers and a detergent-disinfectant diluted with tap water had contaminated perfusion bottles.
TL;DR: Two phenol oxidases, a tyrosinase and a laccase were isolated from cell extracts of the soil bacterium Pseudomonas putida F6 and were stable over a broad range of temperatures and were most stable at 30 °C.
TL;DR: It is proposed that CV degradation occurs via a stepwise demethylation process to yield mono-, di-, tri-, tetra-, penta- and hexa-demethylated CV species.
Abstract: Crystal violet (CV), which has been extensively used as a biological stain and a commercial textile dye, is a recalcitrant molecule. A strain of Pseudomonas putida was isolated that effectively degraded CV: up to 80% of 60 μM CV as the sole carbon source, was degraded in liquid media within 1 week. Nine degradation products were isolated and identified. We propose that CV degradation occurs via a stepwise demethylation process to yield mono-, di-, tri-, tetra-, penta- and hexa-demethylated CV species.
TL;DR: In field experiments neither natural attenuation nor bioaugmentation with P. putida JLR11 affected TNT levels to a significant degree, but the extractable TNT content in rhizosphere soil associated to maize roots decreased by more than 96% in 60 days regardless of inoculation.
Abstract: In situ bioremediation of the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) provides a cost-effective alternative for cleaning up contaminated sites. Here we compare the effectiveness of several bioremediation techniques: natural attenuation, bioaugmentation with TNT-degrading Pseudomonas putida JLR11, phytoremediation with maize (Zea mays L.) and broad beans (Vicia faba L.), and rhizoremediation with maize and broad beans inoculated with P. putida JLR11. Experiments in spiked hydroponic medium demonstrated that inoculation with bacteria did not affect TNT levels. On the other hand, axenic plants were able to remove 32% to 38% of the TNT from the medium. However, when plants were inoculated with bacteria, TNT disappeared to an even greater extent (80% to 88%), a result that advocates a role for P. putida JLR11 in rhizoremediation. In field experiments neither natural attenuation nor bioaugmentation with P. putida JLR11 affected TNT levels to a significant degree. However, the extractable TNT conten...
TL;DR: Biocontrol of the root-knot nematode Meloidogyne javanica was studied on lentil using plant growth-promoting rhizobacteria namely Pseudomonas putida, P. alcaligenes, Paenibacillus polymyxa and Bacillus pumilus and root nodule bacterium Rhizobium sp.
Abstract: Biocontrol of the root-knot nematode Meloidogyne javanica was studied on lentil using plant growth-promoting rhizobacteria (PGPR) namely Pseudomonas putida, P. alcaligenes, Paenibacillus polymyxa and Bacillus pumilus and root nodule bacterium Rhizobium sp. Pseudomonas putida caused greater inhibitory effect on the hatching and penetration of M. javanica followed by P. alcaligenes, P. polymyxa and B. pumilus. Inoculation of any PGPR species alone or together with Rhizobium increased plant growth both in M. javanica-inoculated and -uninoculated plants. Inoculation of Rhizobum caused greater increase in plant growth than caused by any species of plant growth-promoting rhizobacteria in nematode-inoculated plants. Among PGPR, P. putida caused greater increase in plant growth and higher reduction in galling and nematode multiplication followed by P. alcaligenes, P. polymyxa and B. pumilus. Combined use of Rhizobium with any species of PGPR caused higher reduction in galling and nematode multiplication than their individual inoculation. Use of Rhizobium plus P. putida caused maximum reduction in galling and nematode multiplication followed by Rhizobium plus P. alcaligens. Pseudomonas putida caused greater root colonization and siderophore production followed by P. alcaligenes, P. polymyxa and B. pumilus. Analysis of the protein bands of these four species by SDS-PAGE revealed that P. putida had a different protein band profile compared to the protein profiles of P. alcaligenes, P. polymyxa and B. pumilus. However, the protein profiles of P. acaligenes, P. polymyxa and B. pumilus were similar.
Dissertation•
09 Nov 2007
TL;DR: It is found that although the overall number of colony forming units (CFU) did not decline in long-term-starved populations, these populations were very heterogeneous on the plates and contained many dead cells, implying that slow growth of subpopulation of cells at the expenses of dead cells on selective plates might be important for the generation of stationary-phase mutations in P. putida.
Abstract: One of the popular ideas is that decline in methyl-directed mismatch repair (MMR) in carbon-starved bacteriamight facilitate occurrence of stationary-phasemutations.We compared the frequency of accumulation of stationary-phase mutations in carbon-starved Pseudomonas putida wild-type and MMR-defective strains and found that knockout of MMR system increased significantly emergence of base substitutions in starving P. putida. At the same time, the appearance of 1-bp deletion mutations was less affected by MMR in this bacterium. The spectrum of base substitution mutations which occurred in starving populations of P. putida wild-type strain was distinct from mutation spectrum identified in MMR-defective strains. The spectrum of base substitutions differed also in this case when mutants emerged in starved populations of MutS or MutL-defective strains were comparatively analyzed. Based on our results we suppose that othermechanisms thanmalfunctioning ofMMR system in resting cellsmight be considered to explain the accumulation of stationary-phase mutations in P. putida. To further characterize populations of P. putida starved on selective plates, we stained bacteria with LIVE/DEAD kit in situ on agar plates. We found that although the overall number of colony forming units (CFU) did not decline in long-term-starved populations, these populations were very heterogeneous on the plates and contained many dead cells. Our results imply that slow growth of subpopulation of cells at the expenses of dead cells on selective plates might be important for the generation of stationary-phase mutations in P. putida. Additionally, the different survival patterns of P. putida on the same selective plates hint that competitive interactions taking place under conditions of prolonged starvation of microbial populations on semi-solid surfaces might be more complicated than previously assumed. © 2005 Elsevier B.V. All rights reserved. ∗ Corresponding author. Tel.: +372 7 375036; fax: +372 7 420286. E-mail address: maiak@ebc.ee (M. Kivisaar).
TL;DR: The classification results by the proposed method were highly comparable to those based on the DNA gyrase subunit B gene (gyrB) sequence analysis, suggesting the method would be a useful high-throughput method for phylogenetic classification of newly isolated bacteria.
Abstract: A new method for phylogenetic classification of bacterial strains using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is proposed. This method was developed using a bioinformatics-based approach to the rapid identification of bacteria as previously proposed by Demirev and co-workers, which uses ribosomal proteins composed of approximately 50 subunit proteins as biomarkers. Although the amino acid sequences of ribosomal proteins are highly conserved, slight sequence variations can occur at the strain level. Since ribosomal subunit proteins are a complex of housekeeping proteins that have different phylogenetic evolution rates, sequence variation detected as mass differences by MALDI-MS may be useful for the phylogenetic classification of bacteria at strain level. In our proposed method, the first step is the selection of reliable biomarkers through characterization of the expressed ribosomal subunit proteins of a reference strain (usually a genome-sequenced strain) by MALDI-MS. The observed masses in the MALDI mass spectra of cell lysates of sample strains are then compared with the biomarker masses of the reference strain. The biomarkers for each sample strain were designated as present or absent at the reference masses, indicated by 1 or 0, respectively, which were summarized in a table. This table is processed by cluster analysis, generating a phylogenetic tree. In this study, the success of this approach was confirmed by classification of Pseudomonas putida strains because its classification is much more complicated than that of other bacterial strains. Forty-three reliable biomarkers were selected from ribosomal sub-unit proteins of a genome-sequenced strain, P. putida KT2440. The numbers and kinds of biomarkers observed for 16 strains of P. putida, including different biovars, were markedly different, reflecting the variety of the strains. The classification results by the proposed method were highly comparable to those based on the DNA gyrase subunit B gene (gyrB) sequence analysis, suggesting our proposed method would be a useful high-throughput method for phylogenetic classification of newly isolated bacteria.
TL;DR: N-Methylmyosmine, 2,5-dihydroxypyridine and succinic acid were detected and satisfactorily verified for the first time as intermediates of nicotine degradation and an alcohol compound, 1-butanone,4-hydroxy-1-(3-pyridinyl), was found to be a novel product of Nicotine degradation.
Abstract: Nicotine and some related alkaloids in tobacco and tobacco wastes are harmful to health and the environment, and a major environmental requirement is to remove them from tobacco and tobacco wastes. In this study, an isolated strain, S16, identified as Pseudomonas putida biotype A, was used to investigate nicotine degradation. Possible intermediates were identified based on the results of NMR, Fourier-transform (FT)-IR and UV spectroscopy, GC-MS and high-resolution MS (HR-MS) analysis. The pathway of nicotine degradation in P. putida was proposed to be from nicotine to 2,5-dihydroxypyridine through the intermediates N-methylmyosmine, 2'-hydroxynicotine, pseudooxynicotine, 3-pyridinebutanal,C-oxo, 3-succinoylpyridine and 6-hydroxy-3-succinoylpyridine. N-Methylmyosmine, 2,5-dihydroxypyridine and succinic acid were detected and satisfactorily verified for the first time as intermediates of nicotine degradation. In addition, an alcohol compound, 1-butanone,4-hydroxy-1-(3-pyridinyl), was found to be a novel product of nicotine degradation. These findings provide new insights into the microbial metabolism of nicotine and the environmentally friendly route of nicotine degradation.
TL;DR: Results clearly demonstrated that inoculation of the encapsulated bacterial isolates promoted plant growth similar to their respective free cells and could be a novel and feasible technique for application in agricultural industry.
TL;DR: The results show that KT2440 metabolizes glucose and toluene simultaneously, as revealed by net flux analysis of [(13)C]glucose.
Abstract: Pseudomonas putida KT2440(pWW0) can use toluene via the TOL plasmid-encoded catabolic pathways and can use glucose via a series of three peripheral chromosome-encoded routes that convert glucose into 6-phosphogluconate (6PG), namely, the glucokinase pathway, in which glucose is transformed to 6PG through the action of glucokinase and glucose-6-phosphate dehydrogenase. Alternatively, glucose can be oxidized to gluconate, which can be phosphorylated by gluconokinase to 6PG or oxidized to 2-ketogluconate, which, in turn, is converted into 6PG. Our results show that KT2440 metabolizes glucose and toluene simultaneously, as revealed by net flux analysis of [(13)C]glucose. Determination of glucokinase and gluconokinase activities in glucose metabolism, gene expression assays using a fusion of the promoter of the Pu TOL upper pathway to 'lacZ, and global transcriptomic assays revealed simultaneous catabolite repression in the use of these two carbon sources. The effect of toluene on glucose metabolism was directed to the glucokinase branch and did not affect gluconate metabolism. Catabolite repression of the glucokinase pathway and the TOL pathway was triggered by two different catabolite repression systems. Expression from Pu was repressed mainly via PtsN in response to high levels of 2-dehydro-3-deoxygluconate-6-phosphate, whereas repression of the glucokinase pathway was channeled through Crc.
TL;DR: Two different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated soil in Japan, and it was revealed that both plasmids belong to the IncP-9 incompatibility group and their nah upper pathway operons are significantly similar, but not completely identical, to those of pDTG1 and pSLX928-6.
Abstract: Two different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated soil in Japan. One approach was the construction of a broad-host-range cosmid-based metagenomic DNA library, and the other was the so-called exogenous plasmid isolation technique. Our screening of NDO genes in both approaches was based on the functional complementation of Pseudomonas putida strains which contained Tn4655K, a transposon carrying the entire set of naphthalene-catabolic (nah) genes but lacking the NDO-encoding gene. We obtained in the former approach a cosmid clone (pSLX928-6) that carried an nah upper pathway operon for conversion of naphthalene to salicylate, and this operon showed a significantly high level of similarity to the corresponding operon on an IncP-9 naphthalene-catabolic plasmid, pDTG1. In the latter approach, the microbial fraction from the soil was mated with a plasmid-free P. putida strain containing a chromosomal copy of Tn4655K, and transconjugants were obtained that received either a 200- or 80-kb plasmid containing all the nah genes for the complete degradation of naphthalene. Subsequent analysis revealed that (1) both plasmids belong to the IncP-9 incompatibility group; (2) their nah upper pathway operons are significantly similar, but not completely identical, to those of pDTG1 and pSLX928-6; and (3) these plasmids carried genes for the salicylate metabolism by the meta-cleavage pathway.
TL;DR: The cls mutant was more sensitive to toluene shocks and to several antibiotics than the parental strain, suggesting either that the RND efflux pumps involved in the extrusion of these drugs were not working efficiently or that membrane permeability was altered in the mutant.
Abstract: Biological membranes have evolved different mechanisms to modify their composition in response to chemical stimuli in a process called 'homeoviscous adaptation'. Among these mechanisms, modifications in the ratio of saturated/unsaturated fatty acids and in cis/trans fatty acid isomers, cyclopropanation and changes in the phospholipids head group composition have been observed. To further understand the role of phospholipid head groups in solvent stress adaptation, we knocked out the cls (cardiolipin synthase) gene in Pseudomonas putida DOT-T1E. As expected, cls mutant membranes contained less cardiolipin than those of the wild-type strain. Although no significant growth rate defect was observed in the cls mutant compared with the wild-type strain, mutant cells were significantly smaller than the wild-type cells. The cls mutant was more sensitive to toluene shocks and to several antibiotics than the parental strain, suggesting either that the RND efflux pumps involved in the extrusion of these drugs were not working efficiently or that membrane permeability was altered in the mutant. Membranes of the cls mutant strain seemed to be more rigid than those of the parental strain, as observed by measurements of fluorescence polarization using the DPH probe, which intercalates into the membranes. Ethidium bromide is pumped out in Pseudomonas putida by at least one RND efflux pump involved in antibiotic and solvent resistance, and the higher rate of accumulation of ethidium bromide inside mutant cells indicated that functioning of the efflux pumps was compromised as a consequence of the alteration in phospholipid head group composition.
TL;DR: A 2-fold increase of the specific p-hydroxybenzoate production rate (q(p)) was observed when l-tyrosine was supplied to a steady-state C-limited chemostat culture of P. putida S12palB1, indicating that Pal activity is the limiting factor when sufficient l-TYrosine is available.
TL;DR: It is suggested that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.
TL;DR: The mcl-PHA containing high HTD monomer contents was found to have both higher crystallinity and improved tensile strength compared with that of typical mcl -PHA.
Abstract: Medium-chain-length polyhydroxyalkanoates (mcl-PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate, 3-hydroxydodecanoate, and high-content 3-hydroxytetradecanoate (HTD) was produced by knockout mutant Pseudomonas putida KT2442 termed P. putida KTOY06. When grown on 6 to14 g/L single-carbon-source tetradecanoic acid, P. putida KTOY06, which β-oxidation pathway was weakened by deleting genes of 3-ketoacyl-coenzyme A (CoA) thiolase (fadA) and 3-hydroxyacyl-CoA dehydrogenase (fadB), for the first time, produced several mcl-PHA including 31 to 49 mol% HTD as a major monomer. HHx contents in these mcl-PHAs remained approximately constant at less than 3 mol%. In addition, large amounts of oligo-HTD were detected in cells, indicating the limited ability of P. putida KTOY06 in polymerizing long-chain-length 3-hydroxyalkanoates. The mcl-PHA containing high HTD monomer contents was found to have both higher crystallinity and improved tensile strength compared with that of typical mcl-PHA.
TL;DR: A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite l-tyrosine and showed dramatically enhanced production characteristics with controlled l-phenylalanine feeding.
Abstract: A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite l-tyrosine. Efficient production was hampered by product degradation, limited cellular L-tyrosine availability, and formation of the by-product cinnamate via L-phenylalanine. The production host was optimized by inactivation of fcs, the gene encoding the first enzyme in the p-coumarate degradation pathway in P. putida, followed by construction of a phenylalanine-auxotrophic mutant. These steps resulted in a P. putida S12 strain that showed dramatically enhanced production characteristics with controlled l-phenylalanine feeding. During fed-batch cultivation, 10 mM (1.7 g l-1) of p-coumarate was produced from glucose with a yield of 3.8 Cmol% and a molar ratio of p-coumarate to cinnamate of 85:1. © 2006 Springer-Verlag.
TL;DR: The aim of this study was to develop an efficient protocol for the isolation of bacteria, which controls TFRR based on selection of enhanced competitive root‐colonizing bacteria from total rhizosphere soil samples.
Abstract: Aims: Tomato foot and root rot (TFRR), caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl), is an economically important disease of tomato. The aim of this study was to develop an efficient protocol for the isolation of bacteria, which controls TFRR based on selection of enhanced competitive rootcolonizing bacteria from total rhizosphere soil samples. Methods and Results: A total of 216 potentially enhanced bacterial strains were isolated from 17 rhizosphere soil samples after applying a procedure to enrich for enhanced root tip colonizers. Amplified ribosomal DNA restriction analysis, in combination with determination of phenotypic traits, was introduced to evaluate the presence of siblings. One hundred sixteen strains were discarded as siblings. Thirty-eight strains were discarded as potential pathogens based on the sequence of their 16S rDNA. Of the remaining strains, 24 performed equally well or better than the good root colonizer Pseudomonas fluorescens WCS365 in a competitive tomato root tip colonization assay. Finally, these enhanced colonizers were tested for their ability to control TFRR in stonewool, which resulted in seven new biocontrol strains. Conclusions: The new biocontrol strains, six Gram-negative and one Grampositive bacteria, were identified as three Pseudomonas putida strains and one strain each of Delftia tsuruhatensis, Pseudomonas chlororaphis, Pseudomonas rhodesiae and Paenibacillus amylolyticus. Significance and Impact of the Study: We describe a fast method for the isolation of bacteria able to suppress TFRR in stonewool, an industrial plant growth substrate. The procedure minimizes the laborious screens that are a common feature in the isolation of biocontrol strains.
TL;DR: It is shown here that succinyl-CoA is one of the final products of this pathway in Pseudomonas putida and Escherichia coli and in vivo and in vitro studies revealed that the paaE gene encodes the beta-ketoadipyl- CoA thiolase that catalyses the last step of the PA catabolic pathway.
Abstract: Phenylacetic acid (PA) degradation in bacteria involves an aerobic hybrid pathway encoded by the paa gene cluster. It is shown here that succinyl-CoA is one of the final products of this pathway in Pseudomonas putida and Escherichia coli. Moreover, in vivo and in vitro studies revealed that the paaE gene encodes the β-ketoadipyl-CoA thiolase that catalyses the last step of the PA catabolic pathway, i.e. the thiolytic cleavage of β-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA. Succinyl-CoA is suggested as a common final product of aerobic hybrid pathways devoted to the catabolism of aromatic compounds.