Showing papers on "Pseudomonas putida published in 2008"

Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology.

Abstract: A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, 13C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype–phenotype relationships and provides a sound framework to explore this versatile bacterium and to capitalize on its vast biotechnological potential.

A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory.

Abstract: Background Pseudomonas putida is the best studied pollutant degradative bacteria and is harnessed by industrial biotechnology to synthesize fine chemicals. Since the publication of P. putida KT2440's genome, some in silico analyses of its metabolic and biotechnology capacities have been published. However, global understanding of the capabilities of P. putida KT2440 requires the construction of a metabolic model that enables the integration of classical experimental data along with genomic and high-throughput data. The constraint-based reconstruction and analysis (COBRA) approach has been successfully used to build and analyze in silico genome-scale metabolic reconstructions.

Deciphering the genetic determinants for aerobic nicotinic acid degradation: The nic cluster from Pseudomonas putida KT2440

Abstract: The aerobic catabolism of nicotinic acid (NA) is considered a model system for degradation of N-heterocyclic aromatic compounds, some of which are major environmental pollutants; however, the complete set of genes as well as the structural-functional relationships of most of the enzymes involved in this process are still unknown. We have characterized a gene cluster (nic genes) from Pseudomonas putida KT2440 responsible for the aerobic NA degradation in this bacterium and when expressed in heterologous hosts. The biochemistry of the NA degradation through the formation of 2,5-dihydroxypyridine and maleamic acid has been revisited, and some gene products become the prototype of new types of enzymes with unprecedented molecular architectures. Thus, the initial hydroxylation of NA is catalyzed by a two-component hydroxylase (NicAB) that constitutes the first member of the xanthine dehydrogenase family whose electron transport chain to molecular oxygen includes a cytochrome c domain. The Fe(2+)-dependent dioxygenase (NicX) converts 2,5-dihydroxypyridine into N-formylmaleamic acid, and it becomes the founding member of a new family of extradiol ring-cleavage dioxygenases. Further conversion of N-formylmaleamic acid to formic and maleamic acid is catalyzed by the NicD protein, the only deformylase described so far whose catalytic triad is similar to that of some members of the alpha/beta-hydrolase fold superfamily. This work allows exploration of the existence of orthologous gene clusters in saprophytic bacteria and some pathogens, where they might stimulate studies on their role in virulence, and it provides a framework to develop new biotechnological processes for detoxification/biotransformation of N-heterocyclic aromatic compounds.

Evaluation of toluene degradation pathways by two-dimensional stable isotope fractionation.

Abstract: Toluene degradation by several pure and mixed microbial cultures was investigated by two-dimensional compound specific isotope analysis (2D-CSIA) For most of the cultures, the respective toluene degradation pathway and toluene attacking enzymatic step was known The slope of the linear regression for hydrogen (Δδ2H) vs carbon (Δδ13C) discrimination (Λ = Δδ2H/Δδ13C ≈ eHbulk/eCbulk) was determined in order to characterize aerobic and anaerobic toluene degradation pathways The highest Λ value was estimated for the monohydroxylation of the methyl group by Pseudomonas putida (Λ = 53 ± 5) The lowest value was observed for Rhodococcus opacus (Λ = 2 ± 2) due to its insignificant hydrogen fractionation, which indicates that a ring dioxygenase was responsible for the initial attack of toluene The fungus Cladosprium sphaerospermum containing a cytochrome P450-dependent methyl monooxygenase grouped within these extreme values (Λ = 16 ± 6) Λ values for organisms attacking toluene under anoxic conditions by benzyl

Effect of high temperature on Pseudomonas putida NBRI0987 biofilm formation and expression of stress sigma factor RpoS.

Abstract: Pseudomonas is an efficient plant growth–promoting rhizobacteria; however, among the limiting factors for its commercialization, tolerance for high temperature is the most critical one. After screening 2,500 Pseudomnas sp. strains, a high temperature tolerant–strain Pseudomonas putida NBRI0987 was isolated from the drought-exposed rhizosphere of chickpea (Cicer arietinum L. cv. Radhey), which was grown under rain-fed conditions. P. putida NBRI0987 tolerated a temperature of 40°C for ≤ 5 days. To the best of our knowledge, this is the first report of a Pseudomnas sp. demonstrating survival estimated by counting viable cells under such a high temperature. P. putida NBRI0987 colony-forming unit (CFU)/ml on day 10 in both the absence and presence of MgSO4.7H2O (MgSO4) in combination with glycerol at 40°C were 0.0 and 1.7 × 1011, respectively. MgSO4 plus glycerol also enhanced the ability of P. putida NBRI0987 to tolerate high temperatures by inducing its ability to form biofilm. However, production of alginate was not critical for biofilm formation. The present study demonstrates overexpression of stress sigma factor σS (RpoS) when P. putida NBRI0987 is grown under high-temperature stress at 40°C compared with 30°C. We present evidence, albeit indirect, that the adaptation of P. putida NBRI0987 to high temperatures is a complex multilevel regulatory process in which many different genes can be involved.

Interplay of Different Transporters in the Mediation of Divalent Heavy Metal Resistance in Pseudomonas putida KT2440

Abstract: According to in silico analysis, the genome of Pseudomonas putida KT2440 encodes at least four Zn/Cd/Pb efflux transporters—two P-type ATPases (CadA1 and CadA2) and two czc chemiosmotic transporters (CzcCBA1 and CzcCBA2). In this study we showed that all these transporters are functional, but under laboratory conditions only two of them were involved in the mediation of heavy metal resistance in P. putida KT2440. CadA2 conferred Cd2+ and Pb2+ resistance, whereas CzcCBA1 was involved in export of Zn2+, Cd2+, and possibly Pb2+. CadA1, although nonfunctional in P. putida, improved Zn2+ resistance and slightly improved Cd2+ resistance when it was expressed in Escherichia coli. CzcCBA2 contributed to Zn resistance of a czcA1-defective P. putida strain or when the CzcA2 subunit was overexpressed in a transporter-deficient strain. It seemed that CzcA2 could complex with CzcC1 and CzcB1 subunits and therefore complement the loss of CzcA1. The CzcCBA2 transporter itself, however, did not function. Expression of cadA1, cadA2, and czcCBA1 was induced by heavy metals, and the expression levels were dependent on the growth medium and growth phase. Expression of cadA2 and czcCBA1 was nonspecific; both genes were induced by Zn2+, Cd2+, Pb2+, Ni2+, Co2+, and Hg2+. On the other hand, remarkably, expression of cadA1 was induced only by Zn2+. Possible roles of distinct but simultaneously functioning transporters are discussed.

Different, overlapping mechanisms for colonization of abiotic and plant surfaces by Pseudomonas putida.

Abstract: Mechanisms governing biofilm formation have generated considerable interest in recent years, yet comparative analyses of processes for bacterial establishment on abiotic and biotic surfaces are still limited. In this report we have expanded previous information on the genetic determinants required for colonization of plant surfaces by Pseudomonas putida populations and analyzed their correlation with biofilm formation processes on abiotic surfaces. Insertional mutations affecting flagellar genes or the synthesis and transport of the large adhesin LapA lead to decreased adhesion to seeds and biofilm formation on abiotic surfaces. The latter also causes reduced fitness in the rhizosphere. Decreased seed adhesion and altered biofilm formation kinetics are observed in mutants affected in heme biosynthesis and a gene that might participate in oxidative stress responses, whereas a mutant in a gene involved in cytochrome oxidase assembly is affected in the bacterium–plant interaction but not in bacterial establishment on abiotic surfaces. Finally, a mutant altered in lipopolysaccharide biosynthesis is impaired in seed and root colonization but seems to initiate attachment to plastic faster than the wild type. This variety of phenotypes reflects the complexity of bacterial adaptation to sessile life, and the partial overlap between mechanisms leading to biofilm formation on abiotic and biotic surfaces.

Engineering Pseudomonas putida S12 for efficient utilization of D-xylose and L-arabinose

Abstract: The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to utilize xylose as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli. The initial yield on xylose was low (9% [g CDW g substrate−1], where CDW is cell dry weight), and the growth rate was poor (0.01 h−1). The main cause of the low yield was the oxidation of xylose into the dead-end product xylonate by endogenous glucose dehydrogenase (Gcd). Subjecting the XylAB-expressing P. putida S12 to laboratory evolution yielded a strain that efficiently utilized xylose (yield, 52% [g CDW g xylose−1]) at a considerably improved growth rate (0.35 h−1). The high yield could be attributed in part to Gcd inactivity, whereas the improved growth rate may be connected to alterations in the primary metabolism. Surprisingly, without any further engineering, the evolved d-xylose-utilizing strain metabolized l-arabinose as efficiently as d-xylose. Furthermore, despite the loss of Gcd activity, the ability to utilize glucose was not affected. Thus, a P. putida S12-derived strain was obtained that efficiently utilizes the three main sugars present in lignocellulosic hydrolysate: glucose, xylose, and arabinose. This strain will form the basis for a platform host for the efficient production of biochemicals from renewable feedstock.

Transcriptome Analysis of Pseudomonas putida in Response to Nitrogen Availability

Abstract: This work describes a regulatory network of Pseudomonas putida controlled in response to nitrogen availability. We define NtrC as the master nitrogen regulator and suggest that it not only activates pathways for the assimilation of alternative nitrogen sources but also represses carbon catabolism under nitrogen-limited conditions, possibly to prevent excessive carbon and energy flow in the cell.

A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate

Abstract: Pseudomonas putida KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways The genes for these convergent pathways are clustered in three independent regions on the host chromosome A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GltR-2 In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and gap-1, which encodes glyceraldehyde-3-phosphate dehydrogenase GltR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the gtsABCD operon for glucose transport through the inner membrane GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism

Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes

Abstract: Although different biological approaches for detection of anti-personnel mines and other unexploded ordnance (UXO) have been entertained, none of them has been rigorously documented thus far in the scientific literature. The industrial 2,4,6 trinitrotoluene (TNT) habitually employed in the manufacturing of mines is at all times tainted with a small but significant proportion of the more volatile 2,4 dinitrotoluene (2,4 DNT) and other nitroaromatic compounds. By using mutation-prone PCR and DNA sequence shuffling we have evolved in vitro and selected in vivo variants of the effector recognition domain of the toluene-responsive XylR regulator of the soil bacterium Pseudomonas putida that responds to mono-, bi- and trinitro substituted toluenes. Re-introduction of such variants in P. putida settled the transcriptional activity of the cognate promoters (Po and Pu) as a function of the presence of nitrotoluenes in the medium. When strains bearing transcriptional fusions to reporters with an optical output (luxAB, GFP) were spread on soil spotted with nitrotoluenes, the signal triggered by promoter activation allowed localization of the target compounds on the soil surface. Our data provide a proof of concept that non-natural transcription factors evolved to respond to nitroaromatics can be engineered in soil bacteria and inoculated on a target site to pinpoint the presence of explosives. This approach thus opens new ways to tackle this gigantic humanitarian problem.

Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes

Abstract: The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.

Pseudomonas syringae BetT Is a Low-Affinity Choline Transporter That Is Responsible for Superior Osmoprotection by Choline over Glycine Betaine

Abstract: The plant pathogen Pseudomonas syringae derives better osmoprotection from choline than from glycine betaine, unlike most bacteria that have been characterized. In this report, we identified a betaine/carnitine/choline family transporter (BCCT) in P. syringae pv. tomato strain DC3000 that mediates the transport of choline and acetylcholine. This transporter has a particularly low affinity (Km of 876 μM) and high capacity (Vmax of 80 nmol/min/mg of protein) for choline transport relative to other known BCCTs. Although BetT activity increased in response to hyperosmolarity, BetT mediated significant uptake under low-osmolarity conditions, suggesting a role in transport for both osmoprotection and catabolism. Growth studies with mutants deficient in BetT and other choline transporters demonstrated that BetT was responsible for the superior osmoprotection conferred to P. syringae by choline over glycine betaine when these compounds were provided at high concentrations (>100 μM). These results suggest that P. syringae has evolved to survive in relatively choline-rich habitats, a prediction that is supported by the common association of P. syringae with plants and the widespread production of choline, but genus- and species-specific production of glycine betaine, by plants. Among the three putative BCCT family transporters in Pseudomonas aeruginosa and six in Pseudomonas putida, different transporters were predicted to function based on similarity to Escherichia coli BetT than to P. syringae BetT. Functional P. putida and P. aeruginosa transporters were identified, and their possession of a long C-terminal tail suggested an osmoregulatory function for this tail; this function was confirmed for P. syringae BetT using deletion derivatives.

Physiological responses of Pseudomonas putida to formaldehyde during detoxification.

Abstract: Pseudomonas putida KT2440 exhibits two formaldehyde dehydrogenases and two formate dehydrogenase complexes that allow the strain to stoichiometrically convert formaldehyde into CO(2). The strain tolerated up to 1.5 mM formaldehyde and died in the presence of 10 mM. In the presence of 0.5 mM formaldehyde, a sublethal concentration of this chemical, the growth rate decreased by about 40% with respect to growth in the absence of the toxicant. Transcriptomic analysis revealed that in response to low formaldehyde concentrations, a limited number of genes (52) were upregulated. Based on the function of these genes it seems that sublethal concentrations of HCOH trigger responses to overcome DNA and protein damage, extrude this toxic compound, and detoxify it by converting the chemical to CO(2). In strains bearing mutations of the upregulated genes we analysed growth inhibition by 1.5 mM HCOH and killing rates by 10 mM HCOH. Mutants in the MexEF/OprN efflux pump and in the DNA repair genes recA and uvrB were hypersensitive to 10 mM HCOH, the killing rate being three to four orders of magnitude higher than those in the wild-type strain. Mutants in other upregulated genes died at slightly higher or at similar rates to the parental strain. Regarding growth inhibition, we found that mutants in glutathione biosynthesis, stress response mediated by 2-hydroxy acid dehydrogenases and two efflux pumps of the MSF family were unable to grow in the presence of 1.5 mM HCOH. In an independent screening test we searched for mutants which were hypersensitive to formaldehyde, but whose expression did not change in response to this chemical. Two mutants with insertions in recD and fhdA were found which were unable to grow in the presence of 1.5 mM HCOH. The recD mutant was hypersensitive to 10 mM HCOH and died at a higher rate than the parental strain.

In situ monitoring of the biofilm formation of Pseudomonas putida on hematite using flow-cell ATR-FTIR spectroscopy to investigate the formation of inner-sphere bonds between the bacteria and the mineral

Abstract: In situ flow-cell ATR-FTIR using a hematite-coated germanium crystal was used to investigate the chemical interactions between Pseudomonas putida and hematite in real time, when compared with cells not attached to the mineral surface. ATR-FTIR spectra of bacteria growing on hematite showed a shift in the carboxylate signal when compared to the samples obtained from free cells, indicating a chemical interaction between the carboxylate groups and the Fe metal ions of the hematite surface. Small differences in the polysaccharide and phosphoryl regions of the IR spectra of bacteria attached to hematite were also observed. This work shows how the use of in-situ flow-cell experiments with a mineral-coated germanium crystal allows a better description of the bacterial interactions with minerals in real time, as an initial step to understand the fundamental mechanisms involved in the relationship between bacteria and mineral surfaces.

The coordinate regulation of multiple terminal oxidases by the Pseudomonas putida ANR global regulator

Abstract: Summary Pseudomonas putida KT2440 contains a branched aerobic respiratory chain with multiple terminal oxidases. Their relative proportion varies according to environmental conditions. The role of the oxygen-responsive ANR global regulator on expression of these terminal oxidases was analysed. During exponential growth in a highly aerated complete medium, ANR activated expression of the Cbb3-1 terminal oxidase (equivalent to Pseudomonas aeruginosa Cbb3-2), but had little role on expression of other terminal oxidases. In early stationary phase, or under oxygen limitation, inactivation of the anr gene led to increased expression of the bo3-type cytochrome (Cyo) and cyanide-insensitive (CIO) terminal oxidases, and to a much lower expression of Cbb3-1. DNase I footprints identified ANR binding sites at the promoters for these oxidases. Their location suggests that ANR is a transcriptional activator of Cbb3-1 genes and a repressor of CIO genes, consistent with expression data. ANR binding sites at the promoter for Cyo genes suggests a complex regulation in combination with other factors. Therefore, ANR coordinates expression of Cyo, CIO and Cbb3-1, but does not influence cytochrome aa3 and Cbb3-2 terminal oxidases under the conditions analysed. Functional assays showed that Cyo has a leading role during aerobic exponential growth, while Cbb3-1 becomes very important in stationary phase.

A Novel Gene, Encoding 6-Hydroxy-3-Succinoylpyridine Hydroxylase, Involved in Nicotine Degradation by Pseudomonas putida Strain S16

Abstract: Previous research suggested that Pseudomonas spp. may attack the pyrrolidine ring of nicotine in a way similar to mammalian metabolism, resulting in the formation of pseudooxynicotine, the direct precursor of a potent tobacco-specific lung carcinogen. In addition, the subsequent intermediates, 6-hydroxy-3-succinoylpyridine (HSP) and 2,5-dihydroxypyridine (DHP) in the Pseudomonas nicotine degradation pathway are two important precursors for drug syntheses. However, there is little information on the molecular mechanism for nicotine degradation via the pyrrolidine pathway until now. In this study we cloned and sequenced a 4,879-bp gene cluster involved in nicotine degradation. Intermediates N-methylmyosmine, pseudooxynicotine, 3-succinoylpyridine, HSP, and DHP were identified from resting cell reactions of the transformant containing the gene cluster and shown to be identical to those of the pyrrolidine pathway reported in wild-type strain Pseudomonas putida S16. The gene for 6-hydroxy-3-succinoylpyridine hydroxylase (HSP hydroxylase) catalyzing HSP directly to DHP was cloned, sequenced, and expressed in Escherichia coli, and the purified HSP hydroxylase (38 kDa) is NADH dependent. DNA sequence analysis of this 936-bp fragment reveals that the deduced amino acid shows no similarity with any protein of known function.

Dual regulation of zwf-1 by both 2-keto-3-deoxy-6-phosphogluconate and oxidative stress in Pseudomonas putida.

Abstract: Northern blot analysis and a GFP-based reporter assay showed that zwf-1, which encodes glucose-6-phosphate dehydrogenase, was highly induced when Pseudomonas putida KT2440 was cultured in minimal medium containing glucose or gluconate. However, zwf-1 expression was not detected in the presence of pyruvate or succinate. The use of a knockout mutant of HexR, a putative transcription regulator, resulted in constitutively high expression of zwf-1, regardless of the carbon source. An electrophoretic mobility shift assay showed that HexR protein binds to the zwf-1 promoter region and that HexR binding is inhibited by 2-keto-3-deoxy-6-phosphogluconate (KDPG). Despite the presence of gluconate, the edd mutant (non-KDPG producer) was not able to induce the zwf-1 gene. The eda mutant (KDPG overproducer) featured a constitutively high level of zwf-1 expression. Interestingly, zwf-1 was also highly expressed in the presence of oxidative stress-inducing reagents. The level of zwf-1 induction in wild-type cells undergoing oxidative stress did not differ significantly from that observed with the hexR mutant under normal conditions. Interestingly, the hexR mutant was more tolerant of oxidative stress than the wild-type. Expression of zwf-1 was induced by oxidative stress in the edd mutant. Thus, KDPG, a real inducer of zwf-1 gene expression, was not necessary for oxidative-stress induction. In vitro binding of HexR to its cognate promoter region was diminished by menadione and cumene hydroperoxide. The data suggested that HexR might be a dual-sensing regulator of zwf-1 induction that is able to respond to both KDPG and oxidative stress.