Showing papers on "Pseudomonas putida published in 2011"
TL;DR: The directed edition of the P. putida chromosome shown here not only enhances the amenability of this bacterium to deep genomic engineering, but also validates the corresponding approach for similar handlings of a large variety of Gram-negative microorganisms.
Abstract: Summary
The genome of the soil bacterium Pseudomonas putida strain KT2440 has been erased of various determinants of resistance to antibiotics encoded in its extant chromosome To this end, we employed a coherent genetic platform that allowed the precise deletion of multiple genomic segments in a large variety of Gram-negative bacteria including (but not limited to) P putida The method is based on the obligatory recombination between free-ended homologous DNA sequences that are released as linear fragments generated upon the cleavage of the chromosome with unique I-SceI sites, added to the segment of interest by the vector system Despite the potential for a SOS response brought about by the appearance of double stranded DNA breaks during the process, fluctuation experiments revealed that the procedure did not increase mutation rates – perhaps due to the protection exerted by I-SceI bound to the otherwise naked DNA termini With this tool in hand we made sequential deletions of genes mexC, mexE, ttgA and ampC in the genome of the target bacterium, orthologues of which are known to determine various degrees of antibiotic resistance in diverse microorganisms Inspection of the corresponding phenotypes demonstrated that the efflux pump encoded by ttgA sufficed to endow P putida with a high-level of tolerance to β-lactams, chloramphenicol and quinolones, but had little effect on, eg aminoglycosides Analysis of the mutants revealed also a considerable diversity in the manifestation of the resistance phenotype within the population and suggested a degree of synergism between different pumps The directed edition of the P putida chromosome shown here not only enhances the amenability of this bacterium to deep genomic engineering, but also validates the corresponding approach for similar handlings of a large variety of Gram-negative microorganisms
308 citations
TL;DR: Comparative genomics provided a powerful tool to gain new insights into the adaptation of P. putida to specific lifestyles and environmental niches, and clearly demonstrated that horizontal gene transfer played a key role in this adaptation process.
Abstract: Pseudomonas putida is a gram-negative rod-shaped gammaproteobacterium that is found throughout various environments. Members of the species P. putida show a diverse spectrum of metabolic activities, which is indicative of their adaptation to various niches, which includes the ability to live in soils and sediments contaminated with high concentrations of heavy metals and organic contaminants. Pseudomonas putida strains are also found as plant growth-promoting rhizospheric and endophytic bacteria. The genome sequences of several P. putida species have become available and provide a unique tool to study the specific niche adaptation of the various P. putida strains. In this review, we compare the genomes of four P. putida strains: the rhizospheric strain KT2440, the endophytic strain W619, the aromatic hydrocarbon-degrading strain F1 and the manganese-oxidizing strain GB-1. Comparative genomics provided a powerful tool to gain new insights into the adaptation of P. putida to specific lifestyles and environmental niches, and clearly demonstrated that horizontal gene transfer played a key role in this adaptation process, as many of the niche-specific functions were found to be encoded on clearly defined genomic islands.
297 citations
TL;DR: The metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported, finding a functional alternative to the pathogen P. aeruginosa.
Abstract: Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly. Here, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C10:C10). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/gglucose corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation. A functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.
212 citations
TL;DR: For the first time, it was shown that homopolymer PHD and 3HDD monomers dominating PHA could be synthesized by β-oxidation inhibiting P. putida grown on relevant carbon sources.
180 citations
TL;DR: The results indicated the superior performance by P. putida strain AKMP7 in improving survival and growth of wheat plants under heat stress, suggesting the possible role of microorganisms in mitigating adverse effects of climate changes on crop growth and may lead to development of microbe based climate- ready technology.
Abstract: The present study was carried out to investigate the effect of plant growth promoting thermotolerant Pseudomonas putida strain AKMP7 on the growth of wheat plants to heat stress. The results indicated the superior performance by P. putida strain AKMP7 in improving survival and growth of wheat plants under heat stress. The bacterium significantly increased the root and shoot length, dry biomass, tiller, spike let and grain formation of wheat over uninoculated plants. Inoculation reduced membrane injury and the activity of several antioxidant enzymes such as SOD, APX and CAT under heat stress. Inoculation improved the levels of cellular metabolites like proline, chlorophyll, sugars, starch, amino acids, and proteins compared to uninoculated plants. Scanning electron microscopy studies confirmed the colonization of the organism on the root surface. This result suggests the possible role of microorganisms in mitigating adverse effects of climate changes on crop growth and may lead to development of microbe ba...
169 citations
TL;DR: In vitro degradation of single-species Pseudomonas putida biofilms by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase is investigated, suggesting that the addition of high initial titers of specifically selected phages with a proper EPS depolymersase are crucial criteria in the development of phage therapy.
Abstract: Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage φ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage φ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of φ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (104 and 106 pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and φ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.
147 citations
TL;DR: The derivatives of P. putida KT2442 have been developed into a platform for production of various PHA homopolymers and their thermal stability changed with side chain lengths.
Abstract: Pseudomonas putida KT2442 has been a well- studied producer of medium-chain-length (mcl) polyhy- droxyalkanoate (PHA) copolymers containing C6~C14 monomer units. A mutant was constructed from P. putida KT2442 by deleting its phaG gene encoding R-3-hydrox- yacyl-ACP-CoA transacylase and several other β-oxidation related genes including fadB, fadA, fadB2x, and fadAx. This mutant termed P. putida KTHH03 synthesized mcl homo- polymers including poly(3-hydroxyhexanoate) (PHHx) and poly(3-hydroxyheptanoate) (PHHp), together with a near homopolymer poly(3-hydroxyoctanoate-co-2 mol% 3- hydroxyhexanoate) (PHO*) in presence of hexanoate, heptanoate, and octanoate, respectively. When deleted with its mcl PHA synthase genes phaC1 and phaC2 ,t he recombinant mutant termed P. putida KTHH08 harboring pZWJ4-31 containing PHA synthesis operon phaPCJ from Aeromonas hydrophila 4AK4 accumulated homopolymer poly(3-hydroxyvalerate) (PHV) when valerate was used as carbon source. ThephaC deleted recombinant mutant termed P. putida KTHH06 harboring pBHH01 holding PHA synthase PhbC from Ralstonia eutropha produced homopol- ymers poly(3-hydroxybutyrate) (PHB) and poly(4-hydroxy- butyrate) using γ-butyrolactone was added as precursor. All the homopolymers were physically characterized. Their weight average molecular weights ranged from 1.8×10 5 to 1.6×10 6 , their thermal stability changed with side chain lengths. The derivatives of P. putida KT2442 have been developed into a platform for production of various PHA homopolymers.
133 citations
TL;DR: Investigation of the effect of extracellular polymeric substances (EPS) of Gram-positive Bacillus subtilis and Gram-negative Pseudomonas putida on Cu(II) adsorption indicated that the presence of EPS in a biomass sample significantly enhance Cu( II) Adsorption capacity.
114 citations
TL;DR: The apparent robustness of P. putida metabolism indicates that it possesses a certain buffering capacity and a high flexibility to adapt to and counteract different stresses without showing a distinct phenotype, important for the development of whole-cell redox biocatalytic processes that impose equivalent burdens on the cell metabolism.
Abstract: Adenosine phosphate and NAD cofactors play a vital role in the operation of cell metabolism, and their levels and ratios are carefully regulated in tight ranges. Perturbations of the consumption of these metabolites might have a great impact on cell metabolism and physiology. Here, we investigated the impact of increased ATP hydrolysis and NADH oxidation rates on the metabolism of Pseudomonas putida KT2440 by titration of 2,4-dinitrophenol (DNP) and overproduction of a water-forming NADH oxidase, respectively. Both perturbations resulted in a reduction of the biomass yield and, as a consequence of the uncoupling of catabolic and anabolic activities, in an amplification of the net NADH regeneration rate. However, a stimulation of the specific carbon uptake rate was observed only when P. putida was challenged with very high 2,4-dinitrophenol concentrations and was comparatively unaffected by recombinant NADH oxidase activity. This behavior contrasts with the comparably sensitive performance described, for example, for Escherichia coli or Saccharomyces cerevisiae. The apparent robustness of P. putida metabolism indicates that it possesses a certain buffering capacity and a high flexibility to adapt to and counteract different stresses without showing a distinct phenotype. These findings are important, e.g., for the development of whole-cell redox biocatalytic processes that impose equivalent burdens on the cell metabolism: stoichiometric consumption of (reduced) redox cofactors and increased energy expenditures, due to the toxicity of the biocatalytic compounds.
112 citations
TL;DR: In this paper, a mathematical model was developed to describe the transport of bacteria in the presence of suspended kaolinite clay particles in one-dimensional water-saturated porous media.
Abstract: [1] This study is focused on Pseudomonas putida bacteria transport in porous media in the presence of suspended kaolinite clay particles. Experiments were performed with bacteria and kaolinite particles separately to determine their individual transport characteristics in water-saturated columns packed with glass beads. The results indicated that the mass recovery of bacteria and clay particles decreased as the pore water velocity decreased. Batch experiments were carried out to investigate the attachment of Pseudomonas putida onto kaolinite particles. The attachment process was adequately described by a Langmuir isotherm. Finally, bacteria and kaolinite particles were injected simultaneously into a packed column in order to investigate their cotransport behavior. The experimental data suggested that the presence of clay particles significantly inhibited the transport of bacteria in water-saturated porous media. The observed reduction of Pseudomonas putida recovery in the column outflow was attributed to bacteria attachment onto kaolinite particles, which were retained onto the solid matrix of the column. A mathematical model was developed to describe the transport of bacteria in the presence of suspended clay particles in one-dimensional water-saturated porous media. Model simulations were in good agreement with the experimental results.
95 citations
TL;DR: The block copolymer was shown to have the highest tensile strength and Young’s modulus compared with a randomCopolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratios.
Abstract: Polyhydroxyalkanoate (PHA) synthesis genes phaPCJ(Ac) cloned from Aeromonas caviae were transformed into Pseudomonas putida KTOY06ΔC, a mutant of P. putida KT2442, resulting in the ability of the recombinant P. putida KTOY06ΔC (phaPCJ(A.c)) to produce a short-chain-length and medium-chain-length PHA block copolymer consisting of poly-3-hydroxybutyrate (PHB) as one block and random copolymer of 3-hydroxyvalerate (3HV) and 3-hydroxyheptanoate (3HHp) as another block. The novel block polymer was studied by differential scanning calorimetry (DSC), nuclear magnetic resonance, and rheology measurements. DSC studies showed the polymer to possess two glass transition temperatures (T(g)), one melting temperature (T(m)) and one cool crystallization temperature (T(c)). Rheology studies clearly indicated a polymer chain re-arrangement in the copolymer; these studies confirmed the polymer to be a block copolymer, with over 70 mol% homopolymer (PHB) of 3-hydroxybutyrate (3HB) as one block and around 30 mol% random copolymers of 3HV and 3HHp as the second block. The block copolymer was shown to have the highest tensile strength and Young's modulus compared with a random copolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratio. Compared with other commercially available PHA including PHB, PHBV, PHBHHx, and P3HB4HB, the short-chain- and medium-chain-length block copolymer PHB-b-PHVHHp showed differences in terms of mechanical properties and should draw more attentions from the PHA research community.
TL;DR: The data suggest that Pea and Bcs contribute to biofilm formation and in turn their presence contributes to fitness under water-limiting conditions, but not to the extent of alginate.
Abstract: The composition of the exopolysaccharide matrix of Pseudomonas putida mt2 biofilms is relatively undefined as well as the contributions of each polymer to ecological fitness. Here, we describe the role of two putative exopolysaccharide gene clusters, putida exopolysaccharide A (pea) and bacterial cellulose (bcs) in biofilm formation and stability, rhizosphere colonization and matrix hydration under water-limiting conditions. Our findings suggest that pea is involved in the production of a novel glucose, galactose, and mannose-rich polymer that contributes to cell-cell interactions necessary for pellicle and biofilm formation and stability. In contrast, Bcs plays a minor role in biofilm formation and stability, although it does contribute to rhizosphere colonization based on a competition assay. We show that pea expression is highly induced transiently under water-limiting conditions but only slightly by high osmolarity, as determined by qRT-PCR. In contrast, both forms of water stress highly induced bcs expression. Cells deficient in making one or more exopolysaccharide experienced greater dehydration-mediated cell-envelope stress, leading to increased alginate promoter activity. However, this did not lead to increased exopolysaccharide production, except in bcs or pea mutants unable to produce alginate, indicating that P. putida compensates by producing, presumably more Pea or Bcs exopolysaccharides, to facilitate biofilm hydration. Collectively, the data suggest that Pea and Bcs contribute to biofilm formation and in turn their presence contributes to fitness under water-limiting conditions, but not to the extent of alginate.
TL;DR: Antibiotic susceptibility data of five cases and another previous case showed that patients with bacteremia had a high susceptibility of P. putida to anti-pseudomonal β-lactams, which was good, as 26 (92.9%) of the total 28 cases were cured.
TL;DR: In addition to the discovery of a new LOX gene in tomato, this work is the first to show differential induction of LOX isozymes and a more rapid accumulation of 13-hydroperoxy-octadecatrienoic and13-hydroxy- octadecatrianoic acids in rhizobacteria mediated-induced systemic resistance.
Abstract: Some non-pathogenic rhizobacteria called Plant Growth Promoting Rhizobacteria (PGPR) possess the capacity to induce in plant defense mechanisms effective against pathogens. Precedent studies showed the ability of Pseudomonas putida BTP1 to induce PGPR-mediated resistance, termed ISR (Induced Systemic Resistance), in different plant species. Despite extensive works, molecular defense mechanisms involved in ISR are less well understood that in the case of pathogen induced systemic acquired resistance. We analyzed the activities of phenylalanine ammonia-lyase (PAL) and lipoxygenase (LOX), key enzymes of the phenylpropanoid and oxylipin pathways respectively, in tomato treated or not with P. putida BTP1. The bacterial treatment did not stimulate PAL activity and linoleate-consuming LOX activities. Linolenate-consuming LOX activity, on the contrary, was significantly stimulated in P. putida BTP1-inoculated plants before and two days after infection by B. cinerea. This stimulation is due to the increase of transcription level of two isoforms of LOX: TomLoxD and TomLoxF, a newly identified LOX gene. We showed that recombinant TomLOXF preferentially consumes linolenic acid and produces 13-derivative of fatty acids. After challenging with B. cinerea, the increase of transcription of these two LOX genes and higher linolenic acid-consuming LOX activity were associated with a more rapid accumulation of free 13-hydroperoxy-octadecatrienoic and 13-hydroxy-octadecatrienoic acids, two antifungal oxylipins, in bacterized plants. In addition to the discovery of a new LOX gene in tomato, this work is the first to show differential induction of LOX isozymes and a more rapid accumulation of 13-hydroperoxy-octadecatrienoic and 13-hydroxy-octadecatrienoic acids in rhizobacteria mediated-induced systemic resistance.
TL;DR: The block copolymer with a number average molecular weight of 50000 Da and a polydispersity of 3.1 demonstrated a better yield and tensile strength compared with that of its related randomCopolymer and blend of homopolymers of P3HB and P4HB.
TL;DR: Rup4959 enhanced adhesin LapA-dependent biofilm formation, the development of wrinkly colony morphology, and increased Calcofluor stainable exopolysaccharides (EPS) in multicopy or when overexpressed, which correlated with an increase in the global level of c-di-GMP.
Abstract: GGDEF and EAL/HD-GYP protein domains are responsible for the synthesis and hydrolysis of the bacterial secondary messenger cyclic diguanylate (c-di-GMP) through their diguanylate cyclase and phosphodiesterase activities, respectively. Forty-three genes in Pseudomonas putida KT2440 are putatively involved in the turnover of c-di-GMP. Of them only rup4959 (locus PP4959) encodes a GGDEF/EAL response regulator, which was identified in a genome wide analysis as preferentially induced while this bacterium colonizes roots and adjacent soil areas (the rhizosphere). By using fusions to reporter genes it was confirmed that the rup4959 promoter is active in the rhizosphere and inducible by corn plant root exudates and microaerobiosis. Transcription of rup4959 was strictly dependent on the alternative transcriptional factor σ(S) . The inactivation of the rup4959-4957 operon altered the expression of 22 genes in the rhizosphere and had a negative effect upon oligopeptide utilization and biofilm formation. In multicopy or when overexpressed, rup4959 enhanced adhesin LapA-dependent biofilm formation, the development of wrinkly colony morphology, and increased Calcofluor stainable exopolysaccharides (EPS). Under these conditions the inhibition of swarming motility was total and plant root tip colonization considerably less efficient, whereas swimming was partially diminished. This pleiotropic phenotype, which correlated with an increase in the global level of c-di-GMP, was not acquired with increased levels of Rup4959 catalytic mutant at GGDEF as a proof of this response regulator exhibiting diguanylate cyclase activity. A screen for mutants in putative targets of c-di-GMP led to the identification of a surface polysaccharide specific to KT2440, which is encoded by the genes cluster PP3133-PP3141, as essential for phenotypes associated with increased c-di-GMP. Cellulose and alginate were discarded as the overproduced EPS, and lipopolysaccharide (LPS) core and O-antigen were found to be essential for the development of wrinkly colony morphology.
TL;DR: This work describes the generation of novel PHAs (named PHACOS) with a new monomer composition containing thioester groups in the side chain, which confers new properties and made them suitable for chemical modifications after their biosynthesis.
Abstract: This work describes the generation of novel PHAs (named PHACOS) with a new monomer composition containing thioester groups in the side chain, which confers new properties and made them suitable for chemical modifications after their biosynthesis. We have analyzed the PHACOS production abilities of the wild-type strain Pseudomonas putida KT2442 vs. its derived strain P. putida KT42FadB, mutated in the fadB gene from the central metabolic β-oxidation pathway involved in the synthesis of medium-chain-length PHA (mcl-PHA). Different fermentation strategies based on one- or two-stage cultures have been tested resulting in PHACOS with different monomer composition. Using decanoic acid as inducer of the growth and polymer synthesis and 6-acetylthiohexanoic acid as PHA precursor in a two-stage strategy, the maximum yield was obtained by culturing the strain KT42FadB. Nuclear magnetic resonance and gas chromatography coupled to mass spectrometry showed that polymers obtained from the wild-type and KT42FadB strains, included 6-acetylthio-3-hydroxyhexanoic acid (OH-6ATH) and the shorter derivative 4-acetylthio-3-hydroxybutanoic acid (OH-4ATB) in their composition, although in different ratios. While the polymer obtained from KT42FadB strain contained mainly OH-6ATH monomer units, mcl-PHA produced by the wild-type strain contained OH-6ATH and OH-4ATB. Furthermore, polyesters showed differences in the OH-alkyl derivates moiety. The strain KT42FadB overproduced PHACOS when compared to the production rate of the control strain in one- and two-stage cultures. Thermal properties obtained by differential scanning calorimetry indicated that both polymers have different glass transition temperatures related to their composition.
TL;DR: The result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.
TL;DR: Two bacteria identified as Pseudomonas putida and Acinetobacter rhizosphaerae able to rapidly degrade the organophosphate fenamiphos (FEN) were isolated and exhibited high bioremediation potential against spillage-level concentrations of aged residues of FEN and its oxidized derivatives.
TL;DR: Understanding of the P. putida lifestyle at low temperature is improved, with changes seemed directed towards neutralizing problems created by low temperature, such as increased protein misfolding, the increased stability of DNA/RNA secondary structures, reduced membrane fluidity and a reduced growth rate.
Abstract: In its natural habitats (soil, water and rhizosphere), Pseudomonas putida can suffer frequent and long-term changes in temperature that affect its growth and survival. Pseudomonas putida KT2440, a well-characterized model strain, grows optimally at 30°C but can proliferate at temperatures as low as 4°C. However, little information is available on the physiological changes that occur when P. putida grows at low temperatures. To investigate this area, the transcriptome and proteome profiles of cells exponentially growing in a complex medium at 10°C were compared with those of cells exponentially growing at 30°C. Low temperature modified the expression of at least 266 genes (some 5% of the genome). Many of the genes showing differential expression were involved in energy metabolism or in the transport and binding of substrates, although genes implicated in other cellular functions were also affected. Several changes seemed directed towards neutralizing problems created by low temperature, such as increased protein misfolding, the increased stability of DNA/RNA secondary structures, reduced membrane fluidity and a reduced growth rate. The present results improve our understanding of the P. putida lifestyle at low temperature, which may be relevant for its applications in bioremediation and in promotion of plant growth.
TL;DR: The first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440 is characterized, and GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption.
Abstract: Summary Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building-block for drug development. We have char- acterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440. GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption. The proposed GA degradation via the central intermediate 4-- oxalomesaconic acid (OMA) was revisited and all enzymes involved have been identified. Thus, GalD is the prototype of a new subfamily of isomerases that catalyses a biochemical step that remained unknown, i.e. the tautomerization of the OMAketo generated by the GalA dioxygenase to OMAenol. GalB is the founding member of a new family of zinc-containing hydratases that converts OMAenol into 4-carboxy-4- hydroxy-2-oxoadipic acid (CHA). galC encodes the aldolase catalysing CHA cleavage to pyruvic and oxaloacetic acids. The presence of homologous gal clusters outside the Pseudomonas genus sheds light on the evolution and ecology of the gal genes in GA degraders. The gal genes were used for expanding the metabolic abilities of heterologous hosts towards GA degradation, and for engineering a GA cellular biosensor.
TL;DR: It is suggested that the formation of structured P. putida KT2440 biofilm can occur in the absence of exopolysaccharides; however, exopolySaccharides play a role as structural stabilizers.
Abstract: We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P. putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable exopolysaccharide genes was found to form biofilm with a structure similar to wild-type biofilm, but with a stability lower than that of wild-type biofilm. Based on our data, we suggest that the formation of structured P. putida KT2440 biofilm can occur in the absence of exopolysaccharides; however, exopolysaccharides play a role as structural stabilizers.
TL;DR: The results demonstrate that the intracellular presence of PHA granules confers resistance to cell envelope, and conditions to control the cell autolysis in P. putida BXHL in terms of optimal fermentation, PHA content and PHA recovery have been set up by exploring the sensitivity to detergents, chelating agents and wet biomass solubility in organic solvents such as ethyl acetate.
Abstract: Summary The development of efficient recovery processes is essential to reduce the cost of polyhydroxyalkanoates (PHAs) production. In this work, a programmed self- disruptive Pseudomonas putida BXHL strain, derived from the prototype medium-chain-length PHA pro- ducer bacterium P. putida KT2440, was constructed as proofofconceptforexploringthepossibilitytocontrol and facilitate the release of PHA granules to the extra- cellular medium. The new autolytic cell disruption system is based on two simultaneous strategies: the coordinated action of two proteins from the pneumo- coccal bacteriophage EJ-1, an endolysin (Ejl) and a holin (Ejh), and the mutation of the tolB gene, which exhibits alterations in outer membrane integrity that induce lysis hypersensitivity. The ejl and ejh coding genes were expressed under a XylS/Pm monocopy expression system inserted into the chromo- some of the tolB mutant strain, in the presence of 3-methylbenzoate as inducer molecule. Our results demonstrate that the intracellular presence of PHA granules confers resistance to cell envelope. Condi- tions to control the cell autolysis in P. putida BXHL in terms of optimal fermentation, PHA content and PHA recovery have been set up by exploring the sensitivity to detergents, chelating agents and wet biomass solu- bility in organic solvents such as ethyl acetate.
TL;DR: In vitro experiments show that McpT is methylated by CheR and McPT net methylation was diminished in the presence of hydrocarbons, what influences chemotactic movement towards these chemicals.
Abstract: Bacterial chemotaxis is an adaptive behaviour, which requires sophisticated information-processing capabilities that cause motile bacteria to either move towards or flee from chemicals. Pseudomonas putida DOT-T1E exhibits the capability to move towards different aromatic hydrocarbons present at a wide range of concentrations. The chemotactic response is mediated by the McpT chemoreceptor encoded by the pGRT1 megaplasmid. Two alleles of mcpT are borne on this plasmid and inactivation of either one led to loss of this chemotactic phenotype. Cloning of mcpT into a plasmid complemented not only the mcpT mutants but also its transfer to other Pseudomonas conferred chemotactic response to high concentrations of toluene and other chemicals. Therefore, the phenomenon of chemotaxis towards toxic compounds at high concentrations is gene-dose dependent. In vitro experiments show that McpT is methylated by CheR and McpT net methylation was diminished in the presence of hydrocarbons, what influences chemotactic movement towards these chemicals.
TL;DR: A negative counterselection system for Pseudomonas putida based on uracil phosphoribosyltransferase (UPRTase) and sensitivity against the antimetabolite 5-fluorouracil (5-FU) is developed.
Abstract: We developed a negative counterselection system for Pseudomonas putida based on uracil phosphoribosyltransferase (UPRTase) and sensitivity against the antimetabolite 5-fluorouracil (5-FU). We constructed a P. putida strain that is resistant to 5-FU and constructed vectors for the deletion of the surface adhesion protein gene, the flagellum biosynthesis operon, and two endonuclease genes. The genes were efficiently disrupted and left a markerless chromosomal in-frame deletion.
TL;DR: Type II Pseudomonas PHA synthases 1 (PhaC1s) having mutations in these four sites were able to accept lactyl-CoA as a substrate and supported the synthesis of P(3HB-co-LA) in recombinant E. coli, whereas the wild-type PhaC 1s could not accumulate polymers in detectable levels.
Abstract: Previously, we have developed metabolically engineered Escherichia coli strains capable of producing polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] by employing evolved Clostridium propionicum propionate CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19). Introduction of mutations four sites (E130, S325, S477, and Q481) of PhaC1Ps6-19 have been found to affect the polymer content, lactate mole fraction, and molecular weight of P(3HB-co-LA). In this study, we have further engineered type II Pseudomonas PHA synthases 1 (PhaC1s) from Pseudomonas chlororaphis, Pseudomonas sp. 61-3, Pseudomonas putida KT2440, Pseudomonas resinovorans, and Pseudomonas aeruginosa PAO1 to accept short-chain-length hydroxyacyl-CoAs including lactyl-CoA and 3-hydroxybutyryl-CoA as substrates by site-directed mutagenesis of four sites (E130, S325, S477, and Q481). All PhaC1s having mutations in these four sites were able to accept lactyl-CoA as a substrate and supported the synthesis of P(3HB-co-LA) in recombinant E. coli, whereas the wild-type PhaC1s could not accumulate polymers in detectable levels. The contents, lactate mole fractions, and the molecular weights of P(3HB-co-LA) synthesized by recombinant E. coli varied depending upon the source of the PHA synthase and the mutants used. PLA homopolymer could also be produced at ca. 7 wt.% by employing the several PhaC1 variants containing E130D/S325T/S477G/Q481K quadruple mutations in wild-type E. coli XL1-Blue.
TL;DR: It was demonstrated that the production of aromatics such as p-hydroxybenzoate can be improved by co-feeding different carbon sources via different and partially artificial pathways, which opens new perspectives for the efficient production of (fine) chemicals from renewable feedstocks.
Abstract: The key precursors for p-hydroxybenzoate production by engineered Pseudomonas putida S12 are phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), for which the pentose phosphate (PP) pathway is an important source. Since PP pathway fluxes are typically low in pseudomonads, E4P and PEP availability is a likely bottleneck for aromatics production which may be alleviated by stimulating PP pathway fluxes via co-feeding of pentoses in addition to glucose or glycerol. As P. putida S12 lacks the natural ability to utilize xylose, the xylose isomerase pathway from E. coli was introduced into the p-hydroxybenzoate producing strain P. putida S12palB2. The initially inefficient xylose utilization was improved by evolutionary selection after which the p-hydroxybenzoate production was evaluated. Even without xylose-co-feeding, p-hydroxybenzoate production was improved in the evolved xylose-utilizing strain, which may indicate an intrinsically elevated PP pathway activity. Xylose co-feeding further improved the p-hydroxybenzoate yield when co-fed with either glucose or glycerol, up to 16.3 Cmol% (0.1 g p-hydroxybenzoate/g substrate). The yield improvements were most pronounced with glycerol, which probably related to the availability of the PEP precursor glyceraldehyde-3-phosphate (GAP). Thus, it was demonstrated that the production of aromatics such as p-hydroxybenzoate can be improved by co-feeding different carbon sources via different and partially artificial pathways. Moreover, this approach opens new perspectives for the efficient production of (fine) chemicals from renewable feedstocks such as lignocellulose that typically has a high content of both glucose and xylose and (crude) glycerol.
TL;DR: The optimization of fermentation variables using conventional, statistical approaches and aeration/agitation at fermentor level resulted in ~13.5 folds increase in protease production compared to un-optimized conditions, which is the highest level of thermoalkaline protease reported so far by any psychrotrophic bacterium.
Abstract: Production of alkaline protease from various bacterial strains using statistical methods is customary now-a-days. The present work is first attempt for the production optimization of a solvent stable thermoalkaline protease by a psychrotrophic Pseudomonas putida isolate using conventional, response surface methods, and fermentor level optimization. The pre-screening medium amended with optimized (w/v) 1.0% glucose, 2.0% gelatin and 0.5% yeast extract, produced 278 U protease ml-1 at 72 h incubation. Enzyme production increased to 431 Uml-1 when Mg2+ (0.01%, w/v) was supplemented. Optimization of physical factors further enhanced protease to 514 Uml-1 at pH 9.0, 25°C and 200 rpm within 60 h. The combined effect of conventionally optimized variables (glucose, yeast extract, MgSO4 and pH), thereafter predicted by response surface methodology yielded 617 U protease ml-1 at glucose 1.25% (w/v), yeast extract 0.5% (w/v), MgSO4 0.01% (w/v) and pH 8.8. Bench-scale bioreactor level optimization resulted in enhanced production of 882 U protease ml-1 at 0.8 vvm aeration and 150 rpm agitation during only 48 h incubation. The optimization of fermentation variables using conventional, statistical approaches and aeration/agitation at fermentor level resulted in ~13.5 folds increase (882 Uml-1) in protease production compared to un-optimized conditions (65 Uml-1). This is the highest level of thermoalkaline protease reported so far by any psychrotrophic bacterium.
TL;DR: PHE degradation was reduced, and the percentage of PAH-RHD α transcripts was lower with root exudates than with only PHE, but increased over the incubation, while PAh-R HD α gene copy number was higher.
TL;DR: This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled byQuorum-sensing signals.
Abstract: SsoPox, a bifunctional enzyme with organophosphate hydrolase and N-acyl homoserine lactonase activities from the hyperthermophilic archaeon Sulfolobus solfataricus, was overexpressed and purified from recombinant Pseudomonas putida KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for N-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower Km values for these substrates. The highest specificity constant obtained was for N-3-oxo-decanoyl homoserine lactone (kcat/Km = 5.5 × 103 M−1·s−1), but SsoPox can also degrade N-butyryl homoserine lactone (C4-HSL) and N-oxo-dodecanoyl homoserine lactone (oxo-C12-HSL), which are important for quorum sensing in our Pseudomonas aeruginosa model system. When P. aeruginosa PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C4-HSL- and oxo-C12-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.