Putida Strain Research Articles

Global transcriptional response of solvent-sensitive and solvent-tolerant Pseudomonas putida strains exposed to toluene.

Pseudomonas putida strains are generally recognized as solvent tolerant, exhibiting varied sensitivity to organic solvents. Pan-genome analysis has revealed that 30% of genes belong to the core-genome of Pseudomonas. Accessory and unique genes confer high degree of adaptability and capabilities for the degradation and synthesis of a wide range of chemicals. For the use of these microbes in bioremediation and biocatalysis, it is critical to understand the mechanisms underlying these phenotypic differences. In this study, RNA-seq analysis compared the short- and long-term responses of the toluene-sensitive KT2440 strain and the highly tolerant DOT-T1E strain. The sensitive strain activates a larger number of genes in a higher magnitude than DOT-T1E. This is expected because KT2440 bears one toluene tolerant pump, while DOT-T1E encodes three of these pumps. Both strains activate membrane modifications to reduce toluene membrane permeability. The KT2440 strain activates the TCA cycle to generate energy, while avoiding energy-intensive processes such as flagellar biosynthesis. This suggests that KT2440 responds to toluene by focusing on survival mechanisms. The DOT-T1E strain activates toluene degradation pathways, using toluene as source of energy. Among the unique genes encoded by DOT-T1E is a 70 kb island composed of genes of unknown function induced in response to toluene.

An auxin secreting Pseudomonas putida rhizobacterial strain that negatively impacts water-stress tolerance in Arabidopsis thaliana

Here we report some novel observations in the interaction between the model plant, Arabidopsis thaliana and a Pseudomonas putida strain of rhizobacteria which was observed to release copious amounts of the auxin, indole acetic acid (IAA) under water-stressed conditions. On inoculation with A. thaliana, this strain exhibited a strong inhibitory effect on water-stress tolerance ability of the plant, while promoting plant growth under well-watered conditions. Plants exposed to water-stress with simultaneous P. putida inoculation exhibited enhanced drying and reduced plant water content as opposed to water-stressed plants without PGPR inoculation. We conclude that in the concentration used in the present study, this strain of P. putida enhances the susceptibility of A. thaliana to water-stress.

Elimination of persistent emerging micropollutants in a suspended-bed photocatalytic reactor: influence of operating conditions and combination with aerobic biological treatment.

A larger, lab-scale photocatalytic suspended-bed reactor using TiO2 sol-gel-coated expanded clay granules as a bed material was evaluated for oxidative removal of the persistent pharmaceuticals doxycycline, prednisolone, amoxicillin, and sulfamethizole, as well as their mixture, in ppm concentrations. The photocatalytic degradation potential of drug molecules increases as their adsorption affinity increases towards TiO2-containing coatings. The performance of the photocatalytic reactor in the removal of drugs was improved by optimizing the fluidization process parameters. The reactor operation at high bed loadings is determined by the abrasion resistance of the catalyst coating. The long-term stability of the coated bed was enhanced by optimal loading, achieving a higher removal rate while placing moderate mechanical stress on the coated granules. The photocatalytic pretreatment decreased the toxicity of doxycycline solutions to several bacterial strains, including the environmental bacterium Pseudomonas putida and bacterial strains freshly isolated from the activated sludge. The treatment of doxycycline-containing water with a combination of photocatalytic treatment and bio-oxidation resulted in 98% removal of the target in the bioreactor outlet, with no deterioration in the operation of the biological process.

Biostimulation and microbial community profiling reveal insights on RDX transformation in groundwater.

Hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) is a high explosive released to the environment as a result of weapons manufacturing and testing worldwide. At Los Alamos National Laboratory, the Technical Area (TA) 16 260 Outfall discharged high‐explosives‐bearing water from a high‐explosives‐machining facility to Cañon de Valle during 1951 through 1996. These discharges served as a primary source of high‐explosives and inorganic‐element contamination in the area. Data indicate that springs, surface water, alluvial groundwater, and perched‐intermediate groundwater contain explosive compounds, including RDX (hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine); HMX (octahydro‐1,3,5,7‐tetranitro‐1,3,5,7‐tetrazocine); and TNT (2,4,6‐trinitrotoluene). RDX has been detected in the regional aquifer in several wells, and a corrective measures evaluation is planned to identify remedial alternatives to protect the regional aquifer. Perched‐intermediate groundwater at Technical Area 16 is present at depths from 650 ft to 1200 ft bgs. In this study, we examined the microbial diversity in a monitoring well completed in perched‐intermediate groundwater contaminated by RDX, and examined the response of the microbial population to biostimulation under varying geochemical conditions. Results show that the groundwater microbiome was dominated by Actinobacteria and Proteobacteria. A total of 1,605 operational taxonomic units (OTUs) in 96 bacterial genera were identified. Rhodococcus was the most abundant genus (30.6%) and a total of 46 OTUs were annotated as Rhodococcus. One OTU comprising 25.2% of total sequences was closely related to a RDX ‐degrading strain R. erythropolis HS4. A less abundant OTU from the Pseudomonas family closely related to RDX‐degrading strain P. putida II‐B was also present. Biostimulation significantly enriched Proteobacteria but decreased/eliminated the population of Actinobacteria. Consistent with RDX degradation, the OTU closely related to the RDX‐degrading P. putida strain II‐B was specifically enriched in the RDX‐degrading samples. Analysis of the accumulation of RDX‐degradation products reveals that during active RDX degradation, there is a transient increase in the concentration of the degradation products MNX, DNX, TNX, and NDAB. The accumulation of these degradation products suggests that RDX is degraded via sequential reduction of the nitro functional groups followed by abiotic ring‐cleavage. The results suggest that strict anaerobic conditions are needed to stimulate RDX degradation under the TA‐16 site‐specific conditions.

Draft Genome Sequence of a Multi-Metal Resistant Bacterium Pseudomonas putida ATH-43 Isolated from Greenwich Island, Antarctica.

At low concentrations, heavy metals and metalloids are highly toxic for most microorganisms (Lemire et al., 2013). Over evolution, bacteria have developed several molecular mechanisms in order to cope with heavy metal/metalloid toxicity (Nies, 2000; Lemire et al., 2013). Pseudomonas putida belongs to a group of versatile microorganisms capable to thrive in diverse hostile environments, including multi-metal polluted cold sites (Canovas et al., 2003; Zhang et al., 2012; Moreno and Rojo, 2013). Members of P. putida are largely known for their ability to colonize different kinds of environments and to degrade a vast diversity of toxic organic compounds (Wu et al., 2011). In this context, P. putida ATH-43 was isolated from soil sediments at the “Prat” Chilean military base located in Greenwich Island, Antarctica, and was recognized as a mercury/tellurite resistant bacterium (Rodriguez-Rojas et al., 2015). Interestingly, this strain shows tellurite resistance only when grown in the presence of mercury, suggesting a cross-resistance mechanism. Further experimental evidence revealed that P. putida ATH-43 is highly resistant to other toxicants such as Cd2+, Cu2+, CrO42−, and SeO32−, and several antibiotics including streptomycin, cefotaxime, kanamycin, and chloramphenicol (Rodriguez-Rojas et al., 2015). On the other hand, global distillation and grasshopper effect are of major worldwide concern since they apparently provide an explanation for the rapid occurrence of heavy metal/metalloids contamination in pristine polar environments (Ebinghaus et al., 2002; Macdonald et al., 2005). In this context, the genome sequence of P. putida ATH-43 represents an important information source of genetic resistance determinants to multiple stressors currently affecting the Antarctic ecosystem. In this report we present the first draft genome sequence of a P. putida strain isolated from the Antarctic continent. The shotgun sequencing strategy, assembly, and subsequent annotation showed that the ATH-43 strain possesses a wide spectrum of genetic determinants involved in heavy metal and antibiotic resistance, apparently to cope with extreme oxidative stress conditions. P. putida ATH-43 genome now forms part of the 65 genomes of this species registered at the NCBI database (September, 2016) and it is highly related with the endophytic strain P. putida W619, which is also resistant to several heavy metals. Further characterization of multi-metal resistant psychrotrophic bacteria such as P. putida ATH-43 will be promising to develop novel strategies for heavy metal bioremediation in low temperature environments. All genome data has been submitted to NCBI.

Integrated fertilization systems effects on yield, nodulation state and fatty acids composition of soybean (Glycine max)

Combined application of organic and inorganic fertilizers can play an important role for increasing yield and quality of soybean (Glycine max L.). In order to study of effects of biofertilizers and nitrogen rates on yield, nodulation state and fatty acids composition of soybean, a factorial experiment was conducted based on randomized complete block design with three replications in 2012 and 2013. Factors were different rates of nitrogen fertilizer in four levels (without nitrogen and application 25, 50 and 75 kg urea/ ha) and seed inoculation with as biofertilizers in five levels (without inoculation, seed inoculation with Pseudomonas putida strain 41, P. putida strain 186, Azotobacter chroococcum strain 5 and Bradyrhizobium japonicum). The results showed that maximum of grain yield, plant height, number of filled pods and number of grains per plant were obtained from the highest level of nitrogen fertilizer (75 kg urea/ha) and Rh. inoculation. Furthermore, the highest rate of nitrogen usage (75 kg urea/ha) adversely inhibited nodulation of soybean. Number and dry weight of nodules/plant increased significantly with increasing nitrogen application rates up to 50 kg urea/ha. Seed inoculation with biofertilizers increased oil and protein contents. The maximum oil content was obtained by applying 50 kg urea/ha and seed inoculation with Bradyrhizobium. The saturated fatty acids (palmitic and stearic acids) declined in seed inoculation with Bradyrhizobium than the control, while it was vice versa in unsaturated fatty acids (linoleic, linoneic and oleic acids). Based on the results, it was concluded that application of suitable amounts of nitrogen fertilizer (i.e. between 50 and 75 kg urea/ha) as starter in seed inoculation with Bradyrhizobium japonicum can be recommended for profitable soybean production in the study area.

Effects of biofertilizers and cycocel on some physiological and biochemical traits of wheat (Triticum aestivum L.) under salinity stress

ABSTRACTIn order to study the effects of biofertilizers and cycocel on some physiological and biochemical characteristics of wheat (Triticum aestivum L.) under salinity condition, a factorial experiment was conducted based on randomized complete block design with three replications under greenhouse condition in 2015. Treatments were included salinity in four levels [no salt (control or S0), salinity 30 (S1), 60 (S2) and 90 (S3) mM NaCl equivalent of 2.76, 5.53 and 8.3 dS m−1, respectively], four biofertilizers levels [no biofertilizer (F0), seed inoculation by Azotobacter chrocoocum strain 5 (F1), Pseudomonas putida strain 186 (F2), both inoculation Azotobacter + Pseudomonas (F3)] and three cycocel levels [without cycocel as control (C0), application of 600 (C1) and 1000 (C2) mg L−1]. Results showed that salinity severe stress (90 mM) decreased chlorophyll content, relative water content (RWC), total chlorophyll, photochemical efficiency of PSII and yield of wheat. Whereas, soluble sugars and proline content, electrical conductivity (EC), the activity of catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO) enzymes were increased. Similar results were observed in CAT, POD and PPO activities due to inoculation by biofertilizers and cycocel. Salinity at 30 mM increased the photochemical efficiency of PSII and chlorophyll content in plants grown under biofertilizer and cycocel treatment but with increasing salinity up to 90 mM mentioned parameters were decreased. The highest proline and soluble carbohydrate at all salinity levels were observed in plants treated in the highest cycocel level and Azotobacter+ Pseudomonas application. Generally, it was concluded that biofertilizers and cycocel can be used as a proper tool for increasing wheat yield under salinity condition.

Comparative genomic and functional analyses: unearthing the diversity and specificity of nematicidal factors in Pseudomonas putida strain 1A00316.

We isolated Pseudomonas putida (P. putida) strain 1A00316 from Antarctica. This bacterium has a high efficiency against Meloidogyne incognita (M. incognita) in vitro and under greenhouse conditions. The complete genome of P. putida 1A00316 was sequenced using PacBio single molecule real-time (SMRT) technology. A comparative genomic analysis of 16 Pseudomonas strains revealed that although P. putida 1A00316 belonged to P. putida, it was phenotypically more similar to nematicidal Pseudomonas fluorescens (P. fluorescens) strains. We characterized the diversity and specificity of nematicidal factors in P. putida 1A00316 with comparative genomics and functional analysis, and found that P. putida 1A00316 has diverse nematicidal factors including protein alkaline metalloproteinase AprA and two secondary metabolites, hydrogen cyanide and cyclo-(l-isoleucyl-l-proline). We show for the first time that cyclo-(l-isoleucyl-l-proline) exhibit nematicidal activity in P. putida. Interestingly, our study had not detected common nematicidal factors such as 2,4-diacetylphloroglucinol (2,4-DAPG) and pyrrolnitrin in P. putida 1A00316. The results of the present study reveal the diversity and specificity of nematicidal factors in P. putida strain 1A00316.

Isolation and Characterization of Diazinon Degrading Bacteria from Contaminated Agriculture Soils

Background: Organic agricultural pesticides are so stable in ecosystems and biosphere. These compounds affect soil conditions and agricultural products. The aim of this study was isolation of diazinon-degrading bacteria from Kerman pistachio orchards, Southeastern Iran. Methods: Diazinon-degrading bacteria were enriched in Bushnell-Hass medium. Identification and sequencing of prevalent degrading strains was performed using PCR based on amplifying of 16s rDNA. Results: The soil of pistachio orchard has some degrading bacteria that are suitable for elimination of diazinon from soil and environment. Three diazinon-degrading bacteria strains belong to: Pseudomonas putida strain D3, P. fluorescens strain D1 and Achromobacter piechaudii strain D8. The best degrading strain (D1), up to 100 ppm, illustrated a good growth, whereas more than this concentration, the growth was reduced. The results of Gas-Chromatography (GC) confirmed the decomposition of organic pesticide by degrading-bacteria. Conclusion: The results of Gas-Chromatography (GC) confirmed the decomposition of organic pesticide by degrading-bacteria.

Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

Metabolic analysis of the response of Pseudomonas putida DOT-T1E strains to toluene using Fourier transform infrared spectroscopy and gas chromatography mass spectrometry.

IntroductionAn exceptionally interesting stress response of Pseudomonas putida strains to toxic substances is the induction of efflux pumps that remove toxic chemical substances from the bacterial cell out to the external environment. To exploit these microorganisms to their full potential a deeper understanding of the interactions between the bacteria and organic solvents is required. Thus, this study focuses on investigation of metabolic changes in P. putida upon exposure to toluene.ObjectiveInvestigate observable metabolic alterations during interactions of three strains of P. putida (DOT-T1E, and its mutants DOT-T1E-PS28 and DOT-T1E-18) with the aromatic hydrocarbon toluene.MethodsThe growth profiles were measured by taking optical density (OD) measurement at 660 nm (OD660) at various time points during incubation. For fingerprinting analysis, Fourier-transform infrared (FT-IR) spectroscopy was used to investigate any phenotypic changes resulting from exposure to toluene. Metabolic profiling analysis was performed using gas chromatography-mass spectrometry (GC–MS). Principal component—discriminant function analysis (PC-DFA) was applied to the FT-IR data while multiblock principal component analysis (MB-PCA) and N-way analysis of variance (N-way ANOVA) were applied to the GC–MS data.ResultsThe growth profiles demonstrated the effect of toluene on bacterial cultures and the results suggest that the mutant P. putida DOT-T1E−18 was more sensitive (significantly affected) to toluene compared to the other two strains. PC-DFA on FT-IR data demonstrated the differentiation between different conditions of toluene on bacterial cells, which indicated phenotypic changes associated with the presence of the solvent within the cell. Fifteen metabolites associated with this phenotypic change, in P. putida due to exposure to solvent, were from central metabolic pathways. Investigation of MB-PCA loading plots and N-way ANOVA for condition | strain × time blocking (dosage of toluene) suggested ornithine as the most significant compound that increased upon solvent exposure.ConclusionThe combination of metabolic fingerprinting and profiling with suitable multivariate analysis revealed some interesting leads for understanding the mechanism of Pseudomonas strains response to organic solvent exposure.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-016-1054-1) contains supplementary material, which is available to authorized users.

Genome-wide mapping of transcription start sites yields novel insights into the primary transcriptome of Pseudomonas putida.

The environmental bacterium Pseudomonas putida is an organism endowed with a versatile metabolism and stress tolerance traits that are desirable in an efficient production organism. In this work, differential RNA sequencing was used to investigate the primary transcriptome and RNA regulatory elements of P. putida strain KT2440. A total of 7937 putative transcription start sites (TSSs) were identified, where over two-thirds were located either on the opposite strand or internal to annotated genes. For TSSs associated with mRNAs, sequence analysis revealed a clear Shine-Dalgarno sequence but a lack of conserved overrepresented promoter motifs. These TSSs defined approximately 50 leaderless transcripts and an abundance of mRNAs with long leader regions of which 18 contain RNA regulatory elements from the Rfam database. The thiamine pyrophosphate riboswitch upstream of the thiC gene was examined using an in vivo assay with GFP-fusion vectors and shown to function via a translational repression mechanism. Furthermore, 56 novel intergenic small RNAs and 8 putative actuaton transcripts were detected, as well as 8 novel open reading frames (ORFs). This study illustrates how global mapping of TSSs can yield novel insights into the transcriptional features and RNA output of bacterial genomes.

Proof of concept for the simplified breakdown of cellulose by combining Pseudomonas putida strains with surface displayed thermophilic endocellulase, exocellulase and β-glucosidase.

BackgroundThe production and employment of cellulases still represents an economic bottleneck in the conversion of lignocellulosic biomass to biofuels and other biocommodities. This process could be simplified by displaying the necessary enzymes on a microbial cell surface. Such an approach, however, requires an appropriate host organism which on the one hand can withstand the rough environment coming along with lignocellulose hydrolysis, and on the other hand does not consume the generated glucose so that it remains available for subsequent fermentation steps.ResultsThe robust soil bacterium Pseudomonas putida showed a strongly reduced uptake of glucose above a temperature of 50 °C, while remaining structurally intact hence recyclable, which makes it suitable for cellulose hydrolysis at elevated temperatures. Consequently, three complementary, thermophilic cellulases from Ruminiclostridium thermocellum were displayed on the surface of the bacterium. All three enzymes retained their activity on the cell surface. A mixture of three strains displaying each one of these enzymes was able to synergistically hydrolyze filter paper at 55 °C, producing 20 μg glucose per mL cell suspension in 24 h.ConclusionWe could establish Pseudomonas putida as host for the surface display of cellulases, and provided proof-of-concept for a fast and simple cellulose breakdown process at elevated temperatures. This study opens up new perspectives for the application of P. putida in the production of biofuels and other biotechnological products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0505-8) contains supplementary material, which is available to authorized users.

3-Fluorophenmetrazine, a fluorinated analogue of phenmetrazine: Studies on in vivo metabolism in rat and human, in vitro metabolism in human CYP isoenzymes and microbial biotransformation in Pseudomonas Putida and wastewater using GC and LC coupled to (HR)-MS techniques

Wastewater-based epidemiology (WBE) as means to estimate illicit drug and new psychoactive substance (NPS) consumption with spatial and temporal resolution is gaining increasing attention. In order to evaluate a given NPS using WBE, in vivo metabolism and microbial biotransformation of excretion products and unchanged compounds need evaluation. The aims of this study were to identify in vivo phase I and II metabolites of the NPS 3-fluorophenmetrazine (3-FPM) in human and rat urine and study the in vitro contribution of Cytochrome P450 (CYP) isoenzymes in phase I metabolism. Additionally, to study microbial biotransformation products (MBPs) of 3-FPM from incubations in wastewater and in a wastewater isolated Pseudomonas Putida strain. To these aims gas chromatography and liquid chromatography coupled to mass spectrometry were applied. Metabolites and MBPs were isolated from urine and microbial incubations after solid phase extraction and precipitation with or without enzymatic conjungate cleaving. The main transformation pathways were N-oxidation, aryl hydroxylation and subsequent O-methylation, alkyl hydroxylation, oxidation, and degradation of the ethyl-bridge yielding the O/N-bis-dealkylated metabolite, combinations thereof and further glucuronidation or sulfations. The main excretion products in the human urine sample were the unchanged compound and the N-oxide, and the main MBPs were the N-oxide and hydroxylation with subsequent oxidations on the alpha-methyl position. Based on these findings, the proposed strategy for WBE analysis of 3-FPM is quantitative determination of unchanged 3-FPM together with qualitative verification of a number of selected metabolites to verify consumption and rule out discharge.

Multifunctional Pseudomonasputida strain FBKV2 from arid rhizosphere soil and its growth promotional effects on maize under drought stress

In the present study, rhizobacteria were isolated from the rhizosphere of maize, okra, eggplant, tomato, green gram, peanut and red gram grown in arid and semi-arid regions in India and were screened for drought tolerance in Trypticase soy broth (TSB) supplemented with different concentrations of polyethylene glycol 6000 (PEG 6000). Out of 23 isolates, three could tolerate minimal negative water potential of −1.03MPa and were evaluated for plant growth promoting (PGP) traits under control and drought stress (−1.03MPa) conditions. Pseudomonas spp. strain FBKV2 isolated from eggplant (Solanum melongeana L.) rhizosphere, showed multiple PGP traits under both control and drought stress conditions. The strain was identified as Pseudomonas putida by 16S rRNA sequence analysis and the sequence was submitted to GenBank under the accession number KT311002.1. The strain was evaluated for growth promotion of maize (Zea mays L.) under drought stress. Seedlings inoculated with P. putida strain FBKV2 showed better growth in terms of shoot, root length, and dry biomass. Furthermore, inoculation improved cellular metabolites and stomatal conductance in maize seedlings. Scanning electron microscopy confirmed the colonization of P. putida strain FBKV2 on the root surface of maize seedlings. The present study demonstrates that the isolation of indigenous drought tolerant P. putida strain FBKV2 from stressed ecosystems can be a very useful approach for the development of bio-inoculants for drought stress management in crops.

Metabolic Fingerprinting of Pseudomonas putida DOT-T1E Strains: Understanding the Influence of Divalent Cations in Adaptation Mechanisms Following Exposure to Toluene.

Pseudomonas putida strains can adapt and overcome the activity of toxic organic solvents by the employment of several resistant mechanisms including efflux pumps and modification to lipopolysaccharides (LPS) in their membranes. Divalent cations such as magnesium and calcium play a crucial role in the development of solvent tolerance in bacterial cells. Here, we have used Fourier transform infrared (FT-IR) spectroscopy directly on cells (metabolic fingerprinting) to monitor bacterial response to the absence and presence of toluene, along with the influence of divalent cations present in the growth media. Multivariate analysis of the data using principal component-discriminant function analysis (PC-DFA) showed trends in scores plots, illustrating phenotypic alterations related to the effect of Mg2+, Ca2+ and toluene on cultures. Inspection of PC-DFA loadings plots revealed that several IR spectral regions including lipids, proteins and polysaccharides contribute to the separation in PC-DFA space, thereby indicating large phenotypic response to toluene and these cations. Finally, the saturated fatty acid ratio from the FT-IR spectra showed that upon toluene exposure, the saturated fatty acid ratio was reduced, while it increased in the presence of divalent cations. This study clearly demonstrates that the combination of metabolic fingerprinting with appropriate chemometric analysis can result in practicable knowledge on the responses of important environmental bacteria to external stress from pollutants such as highly toxic organic solvents, and indicates that these changes are manifest in the bacterial cell membrane. Finally, we demonstrate that divalent cations improve solvent tolerance in P. putida DOT‑T1E strains.

Molecular Insights into Toluene Sensing in the TodS/TodT Signal Transduction System

TodS is a sensor kinase that responds to various monoaromatic compounds, which either cause an agonistic or antagonistic effect on phosphorylation of its cognate response regulator TodT, and controls tod operon expression in Pseudomonas putida strains. We describe a molecular sensing mechanism of TodS that is activated in response to toluene. The crystal structures of the TodS Per-Arnt-Sim (PAS) 1 sensor domain (residues 43–164) and its complex with toluene (agonist) or 1,2,4-trimethylbenzene (antagonist) show a typical β2α3β3 PAS fold structure (residues 45–149), forming a hydrophobic ligand-binding site. A signal transfer region (residues 150–163) located immediately after the canonical PAS fold may be intrinsically flexible and disordered in both apo-PAS1 and antagonist-bound forms and dramatically adapt an α-helix upon toluene binding. This structural change in the signal transfer region is proposed to result in signal transmission to activate the TodS/TodT two-component signal transduction system. Site-directed mutagenesis and β-galactosidase assays using a P. putida reporter strain system verified the essential residues involved in ligand sensing and signal transfer and suggest that the Phe46 residue acts as a ligand-specific switch.

Simultaneous removal of phenol and cyanide from aqueous solution by co-culture of strain immobilized onto coconut shell activated carbon

In this study, the simultaneous biosorption and bioaccumulation of phenol and cyanide by co-culture/mixed culture of Pseudomonas putida (MTCC 1144) and Serratia odorifera (MTCC 5700) strains immobilized onto coconut shell activated carbon (CSAC) were investigated. The batch experiments were performed for the elimination of phenol and cyanide from mono and binary aqueous solution using initial concentrations of phenol (100–1,000 mg/L) and cyanide (10–100 mg/L). Under optimum conditions, the CSAC was demonstrated a biosorption capacity 500.55 mg/g of phenol and 2.41 mg/g of cyanide at pH 8, biomass dose 30 g/L and temperature 30°C. In a binary component system among six multicomponent isotherms, extended Freundlich model indicates a better fit for both phenol and cyanide. Moreover, the kinetic experimental results show that both phenol and cyanide were defined by the pseudo-first-order kinetic model. Thermodynamic study was established that the simultaneous biosorption and bioaccumulation of phenol indicates the exothermic nature of the process and cyanide indicates the endothermic nature of the process.

Synthesis of chiral 2-alkanols from n-alkanes by a P. putida whole-cell biocatalyst.

The cytochrome P450 monooxygenase CYP154A8 from Nocardia farcinica was previously found to catalyze hydroxylation of linear alkanes (C7 -C9 ) with a high regio- and stereoselectivity. The objective of this study was to integrate CYP154A8 along with suitable redox partners into a whole-cell system for the production of chiral 2-alkanols starting from alkanes. Both recombinant Escherichia coli and Pseudomonas putida whole-cell biocatalysts tested for this purpose showed the ability to produce chiral alkanols, but a solvent tolerant P. putida strain demonstrated several advantages in the applied biphasic reaction system. The optimized P. putida whole-cell system produced ∼16 mM (S)-2-octanol with 87% ee from octane, which is more than sevenfold higher than the previously described system with isolated enzymes. The achieved enantiopurity of the product could further be increased up to 99% ee by adding an alcohol dehydrogenase (ADH) to the alkane-oxidizing P. putida whole-cell systems. By using this setup for the individual conversions of heptane, octane or nonane, 2.6 mM (S)-2-heptanol with 91% ee, 5.4 mM (S)-2-octanol with 97% ee, or 5.5 mM (S)-2-nonanol with 97% ee were produced, respectively. The achieved concentrations of chiral 2-alkanols are the highest reported for a P450-based whole-cell system so far. Biotechnol. Bioeng. 2016;113: 1845-1852. © 2016 Wiley Periodicals, Inc.

Phytochemical Variations and Enhanced Efficiency of Antioxidant and Antimicrobial Ingredients in Salvia officinalis as Inoculated with Different Rhizobacteria

Plants produce a variety of secondary metabolites to improve their performance upon exposure to pathogens, pests, herbivores, or environmental stresses. Secondary metabolism in plants is, therefore, highly regulated by presence of biotic or abiotic elicitors in the environment. The present research was undertaken to characterize plant growth-promoting attributes of four plant growth-promoting rhizobacteria (PGPR) including two Pseudomonas fluorescens (Pf Ap1, Pf Ap18) and two P. putida (Pp Ap9, Pp Ap14) strains, and to determine their role (individually or in consortium) on growth of Salvia officialis, and biosynthesis of secondary metabolites such as essential oils (EOs), total phenolics, and flavonoids. The antioxidant and antibacterial properties of the extracts and EOs obtained from the inoculated plants were also investigated. The PGPR inoculum was applied to soil, cuttings, and foliage. Results indicated that different PGPR strains varied in their efficiency for production of auxin, siderophore, 1-aminocyclopropane-1-carboxylate deaminase, and phosphate solubilization. All individually inoculated plants had significantly higher shoot and root biomass, leaf P content, EOs yield, total phenolics, and flavonoids content compared to uninoculated control plants. The major constituents of EOs, cis-thujene, camphor, and 1,8-cineol, increased following inoculation with reference PGPRs. Although the extract from all inoculated plants had improved antioxidant activity, it was remarkable for the Pf Ap18 strain, which had the lowest IC50 value across treatments. Antibacterial assay of various EOs and their major constituents against pathogenic bacteria showed that the highest activity was observed against Staphylococcus aureus using EOs of Pp Ap14 source. Based on our findings, we suggest that individual inoculation with effective PGPR strains can substantially improve plant growth and secondary metabolism in S. officinalis plants.