Showing papers on "Pseudomonas putida published in 2012"

Benzoxazinoids in Root Exudates of Maize Attract Pseudomonas putida to the Rhizosphere

Abstract: Benzoxazinoids, such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. In addition to their function in plant defence against pests and diseases above-ground, benzoxazinoids (BXs) have also been implicated in defence below-ground, where they can exert allelochemical or antimicrobial activities. We have studied the impact of BXs on the interaction between maize and Pseudomonas putida KT2440, a competitive coloniser of the maize rhizosphere with plant-beneficial traits. Chromatographic analyses revealed that DIMBOA is the main BX compound in root exudates of maize. In vitro analysis of DIMBOA stability indicated that KT2440 tolerance of DIMBOA is based on metabolism-dependent breakdown of this BX compound. Transcriptome analysis of DIMBOA-exposed P. putida identified increased transcription of genes controlling benzoate catabolism and chemotaxis. Chemotaxis assays confirmed motility of P. putida towards DIMBOA. Moreover, colonisation essays in soil with Green Fluorescent Protein (GFP)-expressing P. putida showed that DIMBOA-producing roots of wild-type maize attract significantly higher numbers of P. putida cells than roots of the DIMBOA-deficient bx1 mutant. Our results demonstrate a central role for DIMBOA as a below-ground semiochemical for recruitment of plant-beneficial rhizobacteria during the relatively young and vulnerable growth stages of maize.

Biodegradation of Low Density Polythene (LDPE) by Pseudomonas Species

Abstract: Low density polythene (LDPE) is the most widely used packaging material primarily because of its excellent mechanical properties, barrier properties against water, light weight, low cost and high energy effectiveness. However, due to its ubiquitous nature, and resistance to biodegradability, the disposal strategies are crucial and need attention. Recently, microorganisms have become the focus of interest for environmental friendly disposal of plastic and polymer-based waste. This manuscript aims to investigate the extent of biodegradability of LDPE by four different strains of Pseudomonas bacteria—Pseudomonas aeruginosa PAO1 (ATCC 15729), Pseudomonas aeruginosa (ATCC 15692), Pseudomonas putida (KT2440 ATCC 47054) and Pseudomonas syringae (DC3000 ATCC 10862). Degradation of LDPE was determined by weight loss of the sample, morphological changes, mechanical and spectroscopic variations. The eluted compounds after degradation were analysed by gas chromatography coupled with mass spectroscopy. Our results show that Pseudomonas spp. can degrade LDPE films.

Membrane Vesicle Formation as a Multiple-Stress Response Mechanism Enhances Pseudomonas putida DOT-T1E Cell Surface Hydrophobicity and Biofilm Formation

Abstract: Among the adaptive responses of bacteria to rapid changes in environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms with which bacteria change their cell surface and membranes in the presence of different environmental stresses have been elucidated. Among these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has attracted particular research interest because of its involvement in pathogenic processes, such as that of Pseudomonas aeruginosa biofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response of Pseudomonas putida DOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long-chain alcohols, under osmotic stress caused by NaCl, in the presence of EDTA, and after heat shock, cells of this strain released MV within 10 min in the presence of a stressor. The MV formed showed similar size and charge properties, as well as comparable compositions of proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity, and an enhanced tendency to form biofilms was demonstrated in this study. Therefore, the release of MV as a stress response could be put in a physiological context.

Biosynthesis and characterization of polyhydroxyalkanoates copolymers produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent.

Abstract: The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW) basis were observed when fatty acids ranging from octanoic acid (C8:0) to oleic acid (C18:1) were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the 1 H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (Td) of 264.6 to 318.8 (60.2) o C, melting temperature (Tm) of 43. (60.2) o C, glass transition temperature (Tg )o f21.0 (60.2) o C and apparent melting enthalpy of fusion (DHf) of 100.9 (60.1) J g 21 .

The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: Highlights from a multi-level omics approach

Abstract: Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures. We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system. This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.

Delay of flower senescence by bacterial endophytes expressing 1-aminocyclopropane-1-carboxylate deaminase

Abstract: Aims The ability of 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant growth-promoting bacterial (PGPB) endophytes Pseudomonas fluorescens YsS6 and Pseudomonas migulae 8R6, their ACC deaminase minus mutants and the rhizospheric plant growth-promoting bacterium Pseudomonas putida UW4 to delay the senescence of mini carnation cut flowers was assessed. Methods and results Fresh cut flowers were incubated with either a bacterial cell suspension, the ethylene precursor ACC, the ethylene inhibitor l-α-(aminoethoxyvinyl)-glycine or 0·85% NaCl at room temperature for 11 days. Levels of flower senescence were recorded every other day. To verify the presence of endophytes inside the plant tissues, scanning electron microscopy was performed. Among all treatments, flowers treated with wild-type ACC deaminase-containing endophytic strains exhibited the most significant delay in flower senescence, while flowers treated with the ACC deaminase minus mutants senesced at a rate similar to the control. Flowers treated with Ps. putida UW4 senesced more rapidly than untreated control flowers. Conclusion The only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity so that it may be concluded that this enzyme is directly responsible for the significant delay in flower senescence. Despite containing ACC deaminase activity, Ps. putida UW4 is not taken up by the cut flowers and therefore has no effect on prolonging their shelf life. Significance and impact of the study The world-wide cut flower industry currently uses expensive and potentially environmentally dangerous chemical inhibitors of ethylene to prolong the shelf life of cut flowers. The use of PGPB endophytes with ACC deaminase activity has the potential to replace the chemicals that are currently used by the cut flower industry.

Characterization of a marine-isolated mercury-resistant Pseudomonas putida strain SP1 and its potential application in marine mercury reduction

Abstract: The Pseudomonas putida strain SP1 was isolated from marine environment and was found to be resistant to 280 μM HgCl2. SP1 was also highly resistant to other metals, including CdCl2, CoCl2, CrCl3, CuCl2, PbCl2, and ZnSO4, and the antibiotics ampicillin (Ap), kanamycin (Kn), chloramphenicol (Cm), and tetracycline (Tc). mer operon, possessed by most mercury-resistant bacteria, and other diverse types of resistant determinants were all located on the bacterial chromosome. Cold vapor atomic absorption spectrometry and a volatilization test indicated that the isolated P. putida SP1 was able to volatilize almost 100% of the total mercury it was exposed to and could potentially be used for bioremediation in marine environments. The optimal pH for the growth of P. putida SP1 in the presence of HgCl2 and the removal of HgCl2 by P. putida SP1 was between 8.0 and 9.0, whereas the optimal pH for the expression of merA, the mercuric reductase enzyme in mer operon that reduces reactive Hg2+ to volatile and relatively inert monoatomic Hg0 vapor, was around 5.0. LD50 of P. putida SP1 to flounder and turbot was 1.5 × 109 CFU. Biofilm developed by P. putida SP1 was 1- to 3-fold lower than biofilm developed by an aquatic pathogen Pseudomonas fluorescens TSS. The results of this study indicate that P. putida SP1 is a low virulence strain that can potentially be applied in the bioremediation of HgCl2 contamination over a broad range of pH.

Outer Membrane Protein AlkL Boosts Biocatalytic Oxyfunctionalization of Hydrophobic Substrates in Escherichia coli

Abstract: The outer membrane of microbial cells forms an effective barrier for hydrophobic compounds, potentially causing an uptake limitation for hydrophobic substrates. Low bioconversion activities (1.9 U g(cdw)(-1)) have been observed for the ω-oxyfunctionalization of dodecanoic acid methyl ester by recombinant Escherichia coli containing the alkane monooxygenase AlkBGT of Pseudomonas putida GPo1. Using fatty acid methyl ester oxygenation as the model reaction, this study investigated strategies to improve bacterial uptake of hydrophobic substrates. Admixture of surfactants and cosolvents to improve substrate solubilization did not result in increased oxygenation rates. Addition of EDTA increased the initial dodecanoic acid methyl ester oxygenation activity 2.8-fold. The use of recombinant Pseudomonas fluorescens CHA0 instead of E. coli resulted in a similar activity increase. However, substrate mass transfer into cells was still found to be limiting. Remarkably, the coexpression of the alkL gene of P. putida GPo1 encoding an outer membrane protein with so-far-unknown function increased the dodecanoic acid methyl ester oxygenation activity of recombinant E. coli 28-fold. In a two-liquid-phase bioreactor setup, a 62-fold increase to a maximal activity of 87 U g(cdw)(-1) was achieved, enabling the accumulation of high titers of terminally oxyfunctionalized products. Coexpression of alkL also increased oxygenation activities toward the natural AlkBGT substrates octane and nonane, showing for the first time clear evidence for a prominent role of AlkL in alkane degradation. This study demonstrates that AlkL is an efficient tool to boost productivities of whole-cell biotransformations involving hydrophobic aliphatic substrates and thus has potential for broad applicability.

Development of a New Strategy for Production of Medium-Chain-Length Polyhydroxyalkanoates by Recombinant Escherichia coli via Inexpensive Non-Fatty Acid Feedstocks

Abstract: Pseudomonas putida KT2440 is capable of producing medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when grown on unrelated carbon sources during nutrient limitation. Transcription levels of genes putatively involved in PHA biosynthesis were assessed by quantitative real-time PCR (qRT-PCR) in P. putida grown on glycerol as a sole carbon source. The results showed that two genes, phaG and the PP0763 gene, were highly upregulated among genes potentially involved in the biosynthesis of MCL-PHAs from unrelated carbon sources. Previous studies have described phaG as a 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase, and based on homology, the PP0763 gene was predicted to encode a medium-chain-fatty-acid CoA ligase. High expression levels of these genes during PHA production in P. putida led to the hypothesis that these two genes are involved in PHA biosynthesis from non-fatty acid carbon sources, such as glucose and glycerol. The phaG(pp) and PP0763 genes from P. putida were cloned and coexpressed with the engineered Pseudomonas sp. 61-3 PHA synthase gene phaCl (STQK)(ps) in recombinant Escherichia coli. Up to 400 mg liter(-1) MCL-PHAs was successfully produced from glucose. This study has produced the largest amount of MCL-PHAs reported from non-fatty acid carbon sources in recombinant E. coli to date and opens up the possibility of using inexpensive feedstocks to produce MCL-PHA polymers.

Evaluation of plant-growth-promoting activities of rhizobacterium Pseudomonas putida under herbicide stress

Abstract: The aim of this study was to evaluate the effect of selected herbicides (quizalafop-p-ethyl, clodinafop, metribuzin and glyphosate) at one, two and three times the recommended field rates on the plant-growth-promoting (PGP) traits of Pseudomonas putida strain PS9 isolated from the mustard rhizosphere. P. putida strain PS9 was selected for its high herbicide-tolerance and its production of substantial amounts of indole acetic acid, siderophores (salicylic acid and 2,3 dihydroxy benzoic acid) and exo-polysaccharides. It also exhibits HCN and ammonia releasing ability. A progressive, herbicide-concentration-dependent, decline in PGP properties (except exo-polysaccharide production) of P. putida strain PS9 was observed when the strain was grown in the presence of increasing concentrations of each herbicide. Generally, among the selected herbicides, the maximum toxicity to PGP traits of P. putida strain PS9 was shown by quizalafop-p-ethyl at three times the recommended dose. This study concludes that herbicides should be screened before field application for their degree of toxicity to PGP traits of beneficial microorganisms.

Two small RNAs, CrcY and CrcZ, act in concert to sequester the Crc global regulator in Pseudomonas putida, modulating catabolite repression

Abstract: The Crc protein is a translational repressor that recognizes a specific target at some mRNAs, controlling catabolite repression and co-ordinating carbon metabolism in pseudomonads. In Pseudomonas aeruginosa, the levels of free Crc protein are controlled by CrcZ, a sRNA that sequesters Crc, acting as an antagonist. We show that, in Pseudomonas putida, the levels of free Crc are controlled by CrcZ and by a novel 368 nt sRNA named CrcY. CrcZ and CrcY, which contain six potential targets for Crc, were able to bind Crc specifically in vitro. The levels of CrcZ and CrcY were low under conditions generating a strong catabolite repression, and increased strongly when catabolite repression was absent. Deletion of either crcZ or crcY had no effect on catabolite repression, but the simultaneous absence of both sRNAs led to constitutive catabolite repression that compromised growth on some carbon sources. Overproduction of CrcZ or CrcY significantly reduced repression. We propose that CrcZ and CrcY act in concert, sequestering and modulating the levels of free Crc according to metabolic conditions. The CbrA/CbrB two-component system activated crcZ transcription, but had little effect on crcY. CrcY was detected in P. putida, Pseudomonas fluorescens and Pseudomonas syringae, but not in P. aeruginosa.

The polyhydroxyalkanoate metabolism controls carbon and energy spillage in Pseudomonas putida.

Abstract: The synthesis and degradation of polyhydroxyalkanoates (PHAs), the storage polymer of many bacteria, is linked to the operation of central carbon metabolism. To rationalize the impact of PHA accumulation on central carbon metabolism of the prototype bacterium Pseudomonas putida, we have revisited PHA production in quantitative physiology experiments in the wild-type strain vs. a PHA negative mutant growing under low nitrogen conditions. When octanoic acid was used as PHA precursor and as carbon and energy source, we have detected higher intracellular flux via acetyl-CoA in the mutant strain than in the wild type, which correlates with the stimulation of the TCA cycle and glyoxylate shunt observed on the transcriptional level. The mutant defective in carbon and energy storage spills the additional resources, releasing CO(2) instead of generating biomass. Hence, P. putida operates the metabolic network to optimally exploit available resources and channels excess carbon and energy to storage via PHA, without compromising growth. These findings demonstrate that the PHA metabolism plays a critical role in synchronizing global metabolism to availability of resources in PHA-producing microorganisms.

Synthesis of Diblock copolymer poly-3-hydroxybutyrate -block-poly-3-hydroxyhexanoate [PHB-b-PHHx] by a β-oxidation weakened Pseudomonas putida KT2442

Abstract: Block polyhydroxyalkanoates (PHA) were reported to be resistant against polymer aging that negatively affects polymer properties. Recently, more and more attempts have been directed to make PHA block copolymers. Diblock copolymers PHB-b-PHHx consisting of poly-3-hydroxybutyrate (PHB) block covalently bonded with poly-3-hydroxyhexanoate (PHHx) block were for the first time produced successfully by a recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. The chloroform extracted polymers were characterized by nuclear magnetic resonance (NMR), thermo- and mechanical analysis. NMR confirmed the existence of diblock copolymers consisting of 58 mol% PHB as the short chain length block with 42 mol% PHHx as the medium chain length block. The block copolymers had two glass transition temperatures (T g ) at 2.7°C and −16.4°C, one melting temperature (T m ) at 172.1°C and one cool crystallization temperature (T c ) at 69.1°C as revealed by differential scanning calorimetry (DSC), respectively. This is the first microbial short-chain-length (scl) and medium-chain-length (mcl) PHA block copolymer reported. It is possible to produce PHA block copolymers of various kinds using the recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. In comparison to a random copolymer poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P(HB-co-HHx)) and a blend sample of PHB and PHHx, the PHB-b-PHHx showed improved structural related mechanical properties.

Microbial d-xylonate production

Abstract: d-Xylonic acid is a versatile platform chemical with reported applications as complexing agent or chelator, in dispersal of concrete, and as a precursor for compounds such as co-polyamides, polyesters, hydrogels and 1,2,4-butanetriol. With increasing glucose prices, d-xylonic acid may provide a cheap, non-food derived alternative for gluconic acid, which is widely used (about 80 kton/year) in pharmaceuticals, food products, solvents, adhesives, dyes, paints and polishes. Large-scale production has not been developed, reflecting the current limited market for d-xylonate. d-Xylonic acid occurs naturally, being formed in the first step of oxidative metabolism of d-xylose by some archaea and bacteria via the action of d-xylose or d-glucose dehydrogenases. High extracellular concentrations of d-xylonate have been reported for various bacteria, in particular Gluconobacter oxydans and Pseudomonas putida. High yields of d-xylonate from d-xylose make G. oxydans an attractive choice for biotechnical production. G. oxydans is able to produce d-xylonate directly from plant biomass hydrolysates, but rates and yields are reduced because of sensitivity to hydrolysate inhibitors. Recently, d-xylonate has been produced by the genetically modified bacterium Escherichia coli and yeast Saccharomyces cerevisiae and Kluyveromyces lactis. Expression of NAD+-dependent d-xylose dehydrogenase of Caulobacter crescentus in either E. coli or in a robust, hydrolysate-tolerant, industrial Saccharomyces cerevisiae strain has resulted in d-xylonate titres, which are comparable to those seen with G. oxydans, at a volumetric rate approximately 30 % of that observed with G. oxydans. With further development, genetically modified microbes may soon provide an alternative for production of d-xylonate at industrial scale.

Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440

Abstract: Background Pseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain.

Evaluation of biocontrol potential of Pseudomonas and Bacillus spp. against Fusarium wilt of chickpea

Abstract: In this study, antagonistic effects of 6 isolates of Pseudomonas and 6 isolates of Bacillus genera isolated from rhizosphere of chickpea were evaluated against Fusarium oxysporum f. sp. ciceris as potential biocontrol agents in vitro and in vivo. Fungal inhibition tests were performed using plate assay. Each isolate were tested for the production of protease, siderophore, cyanide hydrogen, indole acetic acid, antifungal volatile and extracellular compound. Twelve isolates were selected according to their high antagonistic efficiency in in vitro which was shown as inhibition zones in the dual-culture assay. According to phenotypic properties, selected isolates were identified as Bacillus subtilis (B1, B6, B28, B40, B99, and B108), Pseudomonas putida (P9 and P10) and P. aeuroginosa (P11, P12, P66 and P112). The ability of bacterial isolates was varied in production of cyanide hydrogen, siderophore, protease and indole acetic acid (IAA). Biocontrol activity and plant growth promotion of bacterial strains were evaluated under greenhouse conditions, in which P. aeuroginosa (P10 and P12), B. subtilis (B1, B6, B28 and B99) and P. aeuroginosa (P12 and B28) provided better control (P <= 0.05) than untreated control (15.8-44.8%) in seed treatment and soil-inoculation, respectively. The growth parameters (plant height, fresh and dry weight of plants) were significantly increased by B28, P12 and P112 isolates in both tests compared to the untreated control. Our results indicate that PGPR improve growth parameters in this plant and can help in the biocontrol of pathogen.

Mechanisms of Resistance to Chloramphenicol in Pseudomonas putida KT2440

Abstract: Pseudomonas putida KT2440 is a chloramphenicol-resistant bacterium that is able to grow in the presence of this antibiotic at a concentration of up to 25 μg/ml. Transcriptomic analyses revealed that the expression profile of 102 genes changed in response to this concentration of chloramphenicol in the culture medium. The genes that showed altered expression include those involved in general metabolism, cellular stress response, gene regulation, efflux pump transporters, and protein biosynthesis. Analysis of a genome-wide collection of mutants showed that survival of a knockout mutant in the TtgABC resistance-nodulation-division (RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline (PQQ) were compromised in the presence of chloramphenicol. The analysis also revealed that an ABC extrusion system (PP2669/PP2668/PP2667) and the AgmR regulator (PP2665) were needed for full resistance toward chloramphenicol. Transcriptional arrays revealed that AgmR controls the expression of the pqq genes and the operon encoding the ABC extrusion pump from the promoter upstream of open reading frame (ORF) PP2669.

Gene expression profiling through microarray analysis in Arabidopsis thaliana colonized by Pseudomonas putida MTCC5279, a plant growth promoting rhizobacterium.

Abstract: Plant growth promotion is a multigenic process under the influence of many factors; therefore an understanding of these processes and the functions regulated may have profound implications. Present study reports microarray analysis of Arabidopsis thaliana plants inoculated with Pseudomonas putida MTCC5279 (MTCC5279) which resulted in significant increase in growth traits as compared with non-inoculated control. The gene expression changes, represented by oligonucleotide array (24652 genes) have been studied to gain insight into MTCC5279 assisted plant growth promotion in Arabidopsis thaliana. MTCC5279 induced upregulated Arabidopsis thaliana genes were found to be involved in maintenance of genome integrity (At5g20850), growth hormone (At3g23890 and At4g36110), amino acid synthesis (At5g63890), abcissic acid (ABA) signaling and ethylene suppression (At2g29090, At5g17850), Ca+2 dependent signaling (At3g57530) and induction of induced systemic resistance (At2g46370, At2g44840). The genes At3g32920 and At2g1...

Ethylene glycol metabolism by Pseudomonas putida.

Abstract: In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.

Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

Abstract: Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTS Fru ), while the other (N-related PTS, or PTS Ntr ) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTS Fru or PTS Ntr genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIA Ntr ) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIA Ntr protein of the PTS Ntr thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. IMPORTANCE This study demonstrates that EIIA Ntr influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIA Ntr protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks.

Novel, Highly Specific N-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids

Abstract: The molecular basis for the ability of bacteria to live on caffeine as a sole carbon and nitrogen source is unknown. Pseudomonas putida CBB5, which grows on several purine alkaloids, metabolizes caffeine and related methylxanthines via sequential N-demethylation to xanthine. Metabolism of caffeine by CBB5 was previously attributed to one broad-specificity methylxanthine N-demethylase composed of two subunits, NdmA and NdmB. Here, we report that NdmA and NdmB are actually two independent Rieske nonheme iron monooxygenases with N(1)- and N(3)-specific N-demethylation activity, respectively. Activity for both enzymes is dependent on electron transfer from NADH via a redox-center-dense Rieske reductase, NdmD. NdmD itself is a novel protein with one Rieske [2Fe-2S] cluster, one plant-type [2Fe-2S] cluster, and one flavin mononucleotide (FMN) per enzyme. All ndm genes are located in a 13.2-kb genomic DNA fragment which also contained a formaldehyde dehydrogenase. ndmA, ndmB, and ndmD were cloned as His(6) fusion genes, expressed in Escherichia coli, and purified using a Ni-NTA column. NdmA-His(6) plus His(6)-NdmD catalyzed N(1)-demethylation of caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively. NdmB-His(6) plus His(6)-NdmD catalyzed N(3)-demethylation of theobromine, 3-methylxanthine, caffeine, and theophylline to 7-methylxanthine, xanthine, paraxanthine, and 1-methylxanthine, respectively. One formaldehyde was produced from each methyl group removed. Activity of an N(7)-specific N-demethylase, NdmC, has been confirmed biochemically. This is the first report of bacterial N-demethylase genes that enable bacteria to live on caffeine. These genes represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste.

Soil Bacterial Consortia and Previous Exposure Enhance the Biodegradation of Sulfonamides from Pig Manure

Abstract: Persistence or degradation of synthetic antibiotics in soil is crucial in assessing their environmental risks. Microbial catabolic activity in a sandy loamy soil with pig manure using 12C- and 14C-labelled sulfamethazine (SMZ) respirometry showed that SMZ was not readily degradable. But after 100 days, degradation in sulfadiazine-exposed manure was 9.2%, far greater than soil and organic manure (0.5% and 0.11%, respectively, p < 0.05). Abiotic degradation was not detected suggesting microbial catabolism as main degradation mechanism. Terminal restriction fragment length polymorphism showed biodiversity increases within 1 day of SMZ spiking and especially after 200 days, although some species plummeted. A clone library from the treatment with highest degradation showed that most bacteria belonged to α, β and γ classes of Proteobacteria, Firmicutes, Bacteroidetes and Acidobacteria. Proteobacteria (α, β and γ), Firmicutes and Bacteroidetes which were the most abundant classes on day 1 also decreased most following prolonged exposure. From the matrix showing the highest degradation rate, 17 SMZ-resistant isolates biodegraded low levels of 14 C-labelled SMZ when each species was incubated separately (0.2–1.5%) but biodegradation was enhanced when the four isolates with the highest biodegradation were incubated in a consortium (Bacillus licheniformis, Pseudomonas putida, Alcaligenes sp. and Aquamicrobium defluvium as per 16S rRNA gene sequencing), removing up to 7.8% of SMZ after 20 days. One of these species (B. licheniformis) was a known livestock and occasional human pathogen. Despite an environmental role of these species in sulfonamide bioremediation, the possibility of horizontal transfer of pathogenicity and resistance genes should caution against an indiscriminate use of these species as sulfonamide degraders.

Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation

Abstract: Genomic analysis of Pseudomonas putida : genes in a genome island are crucial for nicotine degradation

Effects of bacterial ACC deaminase on Brassica napus gene expression.

Abstract: Plants in association with plant growth-promoting rhizobacteria can benefit from lower plant ethylene levels through the action of the bacterial enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. This enzyme cleaves the immediate biosynthetic precursor of ethylene, ACC. Ethylene is responsible for many aspects of plant growth and development but, under stressful conditions, it exacerbates stress symptoms. The ACC deaminase-containing bacterium Pseudomonas putida UW4 is a potent plant growth-promoting strain and, as such, was used to elaborate the detailed role of bacterial ACC deaminase in Brassica napus (canola) plant growth promotion. Transcriptional changes in bacterially treated canola plants were investigated with the use of an Arabidopsis thaliana oligonucleotide microarray. A heterologous approach was necessary because there are few tools available at present to measure global expression changes in nonmodel organisms, specifically with the sensitivity of microarrays. The results indicate...

pH-stat fed-batch process to enhance the production of cis, cis-muconate from benzoate by Pseudomonas putida KT2440-JD1.

Abstract: Pseudomonas putida KT2440-JD1 is able to cometabolize benzoate to cis, cis-muconate in the presence of glucose as growth substrate. P. putida KT2440-JD1 was unable to grow in the presence of concentrations above 50 mM benzoate or 600 mM cis, cis-muconate. The inhibitory effects of both compounds were cumulative. The maximum specific uptake rate of benzoate was higher than the specific production rate of cis, cis-muconate during growth on glucose in the presence of benzoate, indicating that a benzoate derivative accumulated in the cells, which is likely to be catechol. Catechol was shown to reduce the expression level of the ben operon, which encodes the conversion of benzoate to cis, cis-muconate. To prevent overdoses of benzoate, a pH-stat fed-batch process for the production of cis, cis-muconate from benzoate was developed, in which the addition of benzoate was coupled to the acidification of the medium. The maximum specific production rate during the pH-stat fed-batch process was 0.6 g (4.3 mmol) g dry cell weight -1 h -1, whereas 18.5 g L -1 cis, cis-muconate accumulated in the culture medium with a molar product yield of close to 100%. Proteome analysis revealed that the outer membrane protein H1 was upregulated during the pH-stat fed-batch process, whereas the expression of 10 other proteins was reduced. The identified proteins are involved in energy household, transport, translation of RNA, and motility. © 2011 American Institute of Chemical Engineers (AIChE).