Metabolic Engineering of Pseudomonas putida KT2440 for De Novo Biosynthesis of Vanillic Acid.

Vanillic acid (VA), as a plant-derived phenolic acid compound, has widespread applications and good market prospects. However, the traditional production process cannot meet market demand. In this study, Pseudomonas putida KT2440 was used for de novo biosynthesis of VA. Multiple metabolic engineering strategies were applied to construct these P. putida-based cell factories, including the introduction of a Hs-OMTopt, engineering the cofactor S-adenosylmethionine supply pathway through the overexpression of metX and metH, reforming solubility of Hs-OMTopt, increasing a second copy of Hs-OMTopt, and the optimization of the fermentation medium. The resulting strain, XCS17, de novo biosynthesized 5.4 g/L VA from glucose in a fed-batch fermentation system; this is the highest VA production titer reported up to recently. This study showed that P. putida KT2440 is a robust platform for achieving the effective production of phenolic acids.

Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control.

For engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared with normal cellular processes. Within a certain metabolic leeway, this competitive process has no impact on growth. However, once this leeway, or free capacity, is fully utilized, the extra load becomes a metabolic burden that inhibits cellular processes and triggers a broad cellular response, reducing cell growth and often hindering the production of heterologous proteins. In this study, we sought to characterize the metabolic rearrangements occurring in the central metabolism of Pseudomonas putida at different levels of metabolic load. To this end, we constructed a P. putida KT2440 strain that expressed two genes encoding fluorescent proteins, one in the genome under constitutive expression to monitor the free capacity, and the other on an inducible plasmid to probe heterologous protein production. We found that metabolic fluxes are considerably reshuffled, especially at the level of periplasmic pathways, as soon as the metabolic load exceeds the free capacity. Heterologous protein production leads to the decoupling of anabolism and catabolism, resulting in large excess energy production relative to the requirements of protein biosynthesis. Finally, heterologous protein production was found to exert a stronger control on carbon fluxes than on energy fluxes, indicating that the flexible nature of P. putida's central metabolic network is solicited to sustain energy production.

Type VI secretion system drives bacterial diversity and functions in multispecies biofilms.

Type VI secretion system (T6SS) plays an essential role in interspecies interactions and provides an advantage for a strain with T6SS in multispecies biofilms. However, how T6SS drives the bacterial community structure and functions in multispecies biofilms still needs to be determined. Using gene deletion and Illumina sequencing technique, we estimated bacterial community responses in multispecies biofilms to T6SS by introducing T6SS-containing Pseudomonas putida KT2440. Results showed that the niche structure shifts of multispecies biofilms were remarkably higher in the presence of T6SS than in the absence of T6SS. The presence of T6SS significantly drove the variation in microbial composition, reduced the alpha-diversity of bacterial communities in multispecies biofilms, and separately decreased and increased the relative abundance of Proteobacteria and Bacteroidota. Co-occurrence network analysis with inferred putative bacterial interactions indicated that P. putida KT2440 mainly displayed strong negative associations with the genera of Psychrobacter, Cellvibrio, Stenotrophomonas, and Brevundimonas. Moreover, the function redundancy index of the bacterial community was strikingly higher in the presence of T6SS than in the absence of T6SS, regardless of whether relative abundances of bacterial taxa were inhibited or promoted. Remarkably, the increased metabolic network similarity with T6SS-containing P. putida KT2440 could enhance the antibacterial activity of P. putida KT2440 on other bacterial taxa. Our findings extend knowledge of microbial adaptation strategies to potential bacterial weapons and could contribute to predicting biodiversity loss and change in ecological functions caused by T6SS.

Biotransformation Of l-Tryptophan To Produce Arcyriaflavin A With Pseudomonas putida KT2440.

Natural products such as indolocarbazoles are a valuable source of highly bioactive compounds with numerous potential applications in the pharmaceutical industry. Arcyriaflavin A, isolated from marine invertebrates and slime molds, is one representative of this group and acts as a cyclin D1-cyclin-dependent kinase 4 inhibitor. To date, access to this compound has mostly relied on multi-step total synthesis. In this study, biosynthetic access to arcyriaflavin A was explored using recombinant Pseudomonas putida KT2440 based on a previously generated producer strain. We used a Design of Experiment approach to analyze four key parameters, which led to the optimization of the bioprocess. By engineering the formation of outer membrane vesicles and using an adsorbent in the culture broth, we succeeded to increase the yield of arcyriaflavin A in the cell-free supernatant, resulting in a nearly eight-fold increase in the overall production titers. Finally, we managed to scale up the bioprocess leading to a final yield of 4.7 mg arcyriaflavin A product isolated from 1 L of bacterial culture. Thus, this study showcases an integrative approach to improve biotransformation and moreover also provides starting points for further optimization of indolocarbazole production in P. putida.

Assessing the Effectiveness of Eco-Friendly Management Approaches for Controlling Wheat Yellow Rust and Their Impact on Antioxidant Enzymes.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive disease that causes significant yield losses in wheat production worldwide, including in Egypt. The use of biocontrol agents is among the best eco-friendly management strategies to control this disease, as they are more sustainable and environmentally friendly than traditional chemical control methods. In a comparative analysis, antioxidant enzyme activity and various management approaches were compared with two bacterial biocontrol agents, Bacillus subtilis and Pseudomonas putida. This study showed the remarkable efficacy of endophytic bacteria, B. subtilis and P. putida, in mitigating wheat stripe rust infection across three wheat varieties, namely Misr1, Gimmeiza11, and Sids12. B. subtilis exhibited superior performance compared to P. putida, resulting in infection types of 1 and 2.66, respectively, following inoculation. The highest reduction rate was observed with Tilit fungicide (500 ppm), followed by B. subtilis and Salicylic acid (1000 ppm), respectively. Variations in wheat varieties' response to Pst infection were observed, with Misr1 exhibiting the lowest infection and Sids12 showing high susceptibility. Among the tested inducers, Salicylic acid demonstrated the greatest reduction in disease infection, followed by Indole acetic acid, while Oxalic acid exhibited the lowest decrease. Additionally, the study evaluated the activities of five antioxidant enzymes, including Catalase, Ascorbate peroxidase (APX), glutathione reductase (GR), Superoxide dismutase (SOD), and peroxidase (POX), in the wheat-stripe rust interaction under different integrated management approaches. The wheat variety Misr1 treated with Tilit (500 ppm), B. subtilis, Salicylic acid, Montoro (500 ppm), and P. putida exhibited the highest increase in all enzymatic activities. These findings provide valuable insights into the effectiveness of B. subtilis and P. putida as biocontrol agents for wheat stripe rust control in Egypt, emphasizing their potential role in sustainable, integrated, and environmentally friendly management practices.

Exposure to polystyrene nanoplastics reduces bacterial and fungal biomass in microfabricated soil models.

Nanoplastics have been proven to induce toxicity in diverse organisms, yet their effect on soil microbes like bacteria and fungi remains largely unexplored. In this paper, we used micro-engineered soil models to investigate the effect of polystyrene (PS) nanospheres on Pseudomonas putida and Coprinopsis cinerea. Specifically, we explored the effects of increasing concentrations of 60 nm carboxylated bovine serum albumin (BSA) coated nanospheres (0, 0.5, 2, and 10 mg/L) on these bacterial and fungal model organisms respectively, over time. We found that both microorganisms could disperse through the PS solution, but long-distance dispersal was reduced by high concentrations. Microbial biomass decreased in all treatments, in which bacteria showed a linear dose response with the strongest effect at 10 mg/L concentration, and fungi showed a non-linear response with the strongest effect at 2 mg/L concentration. At the highest nanoplastics concentration, the first colonizing fungal hyphae adsorbed most of the PS nanospheres present in their vicinity, in a process that we termed the 'vacuum cleaner effect'. As a result, the toxicity effect of the original treatment on subsequently growing fungal hyphae was reduced to a growth level indistinguishable from the control. We did not find evidence that nanoplastics are able to penetrate bacterial nor fungal cell walls. Overall, our findings provide evidence that nanoplastics can cause a direct negative effect on soil microbes and highlight the need for further studies that can explain how the microbial stress response might affect soil functions.

Impact of Environmental Stresses on the Antibacterial Activity of Graphene Oxide (GO) Nanoparticles against P. putida Biofilms.

As the production of graphene-based nanomaterials such as GO is increasing, it is expected that a large amount of GO waste will be generated. The environment (i.e., soil and aquatic systems) will be amongst the final repositories of these wastes which means important natural microbial communities in such environments will be at risk of GO exposure. However, little is known about how these communities respond to environmental stresses in synergy with the presence of GO. In this study, the effect of three different stress conditions: temperature (5, 25 and 40 °C); pH (5 to 9) and osmotic stress (51, 219 and 320 mM NaCl) in addition to GO treatment was investigated on the viability and physiology of biofilms and planktonic cells of soil bacterium P. putida. It was found that planktonic cells were more resistant to GO alone compared to biofilms. However, the cells were sensitive to GO when exposed to pH or osmotic stresses. Temperature was not found to influence the survival of biofilm with or without exposure to GO. However, low pH caused a reduction in colony-forming units (CFU) at pHs 5 and 6 for the pre-treated samples, while biofilms at pH 7-9 did not show any decrease. Interestingly, the post-treatment of planktonic cells or biofilms with GO showed a significant reduction in CFU at all pH ranges. The effect of higher osmotic stress in combination with GO resulted in a significant reduction in biofilms. These results show that the effect of stresses naturally occurring in the environment can be affected and changed when in combination with GO and can potentially affect the balance of natural biofilms.

Integrated Application of Salicylic Acid and PGPRs to Control Fusarium Wilt of Chickpea.

BACKGROUND: Fusarium wilt and Ascochyta blight are the most important diseases of chickpea. The current study was designed to investigate the individual and combined effect of salicylic acid (SA) with Pseudomonas stutzeri and Pseudomonas putida to suppress Fusarium wilt and promote growth of chickpea varieties: Thal-2006 and Punjab-2008. METHODS: At the time of sowing, inoculum of Fusarium oxysporum was applied to the soil and the incidence of Fusarium wilt was recorded after 60 days. The seeds were inoculated with Pseudomonas stutzeri and Pseudomonas putida prior to sowing. Chickpea plants were treated with salicylic acid at seedling stage. RESULTS: The combination of P. stutzeri and SA significantly increased root length (166% and 145%), shoot height (50% and 47%) and shoot biomass (300% and 233%) in cv. Thal-2006 and cv. Punjab-2008, respectively, in infected plants. Similarly, the combined treatment of P. putida + SA, also enhanced the plant growth parameters of chickpea varieties. Maximum reduction in disease severity was observed in both P. stutzeri + SA (90% and 84%) and P. putida + SA (79% and 77%) treatments in cv. Thal-2006 and Punjab-2008, respectively. Both P. putida + SA and P. stutzeri + SA treatments resulted in increased leaf relative water and total protein content, peroxidase, superoxide dismutase, phenylalanine ammonia-lyase and polyphenol oxidase activities in both resistant (cv. Thal-2006) and susceptible (cv. Punjab-2008) cultivars. Both treatments also significantly reduced malondialdehyde (MDA) and proline content in cv. Thal-2006 and Punjab-2008. Cultivar Thal-2006 was more effective than cv. Punjab-2008. CONCLUSIONS: The results suggested that, in combination, salicylic acid and P. stutzeri may play an important role in controlling Fusarium wilt diseases by inducing systemic resistance in chickpea.

Engineering isoprenoids production in metabolically versatile microbial host Pseudomonas putida.

With the increasing need for microbial bioproduction to replace petrochemicals, it is critical to develop a new industrial microbial workhorse that improves the conversion of lignocellulosic carbon to biofuels and bioproducts in an economically feasible manner. Pseudomonas putida KT2440 is a promising microbial host due to its capability to grow on a broad range of carbon sources and its high tolerance to xenobiotics. In this study, we engineered P. putida KT2440 to produce isoprenoids, a vast category of compounds that provide routes to many petrochemical replacements. A heterologous mevalonate (MVA) pathway was engineered to produce potential biofuels isoprenol (C5) and epi-isozizaene (C15) for the first time in P. putida. We compared the difference between three different isoprenoid pathways in P. putida on isoprenol production and achieved 104 mg/L of isoprenol production in a batch flask experiment through optimization of the strain. As P. putida can natively consume isoprenol, we investigated how to prevent this self-consumption. We discovered that supplementing L-glutamate in the medium can effectively prevent isoprenol consumption in P. putida and metabolomics analysis showed an insufficient energy availability and an imbalanced redox status during isoprenol degradation. We also showed that the engineered P. putida strain can produce isoprenol using aromatic substrates such as p-coumarate as the sole carbon source, and this result demonstrates that P. putida is a valuable microbial chassis for isoprenoids to achieve sustainable biofuel production from lignocellulosic biomass.

The Connection between Czc and Cad Systems Involved in Cadmium Resistance in Pseudomonas putida.

Heavy metal pollution is widespread and persistent, and causes serious harm to the environment. Pseudomonas putida, a representative environmental microorganism, has strong resistance to heavy metals due to its multiple efflux systems. Although the functions of many efflux systems have been well-studied, the relationship between them remains unclear. Here, the relationship between the Czc and Cad systems that are predominantly responsible for cadmium efflux in P. putida KT2440 is identified. The results demonstrated that CzcR3, the response regulator of two-component system CzcRS3 in the Czc system, activates the expression of efflux pump genes czcCBA1 and czcCBA2 by directly binding to their promoters, thereby helping the strain resist cadmium stress. CzcR3 can also bind to its own promoter, but it has only a weak regulatory effect. The high-level expression of czcRS3 needs to be induced by Cd2+, and this relies on the regulation of CadR, a key regulator in the Cad system, which showed affinity to czcRS3 promoter. Our study indicates that the Cad system is involved in the regulation of the Czc system, and this relationship is important for maintaining the considerable resistance to cadmium in P. putida.

Exhaustion of pentachlorophenol in soil microcosms with three Pseudomonas species as detoxification agents.

Pentachlorophenol (PCP) is a toxic compound, which is widely used as a wood preservative product and general biocide. It is persistent in the environment and has been classified as a persistent organic pollutant to be reclaimed in many countries. Bioremediation is an emerging approach to rehabilitating areas polluted by recalcitrant xenobiotics. In the present study, we evaluated the potential of three strains of Pseudomonas (P. putida S121, P. rhizophila S211, and P. fuscovagiceae S115) as bioremediation agents in depletion and detoxification of PCP in soil microcosms. PCP removal was effectively optimized using a central-composite experimental design and response surface methodology (RSM). The optimum conditions for maximum PCP removal yield (85 ± 5%) were: 500 mg/kg PCP concentration, 108 UFC/g soil inoculum size of each strain and 55 days incubation period. The bacterial strains, P. putida, P. rhizophila, and P. fuscovagiceae, showed good capability to tolerate and degrade PCP so that they could be successfully used in synergistic effect to treat PCP polluted soils.

Role of bacterial cell surface sulfhydryl sites in cadmium detoxification by Pseudomonas putida.

Understanding bacterial metal detoxification systems is crucial for determining the environmental impacts of metal pollution and for developing advanced bioremediation and water disinfection strategies. Here, we explore the role of cell surface sulfhydryl sites in bacterial detoxification of Cd, using Pseudomonas putida with surface sulfhydryl sites mostly on its EPS molecules as a model organism. Our results show that 5 and 20 ppm Cd in LB growth medium affects the lag phase of P. putida, but not the overall extent of cell growth at stationary phase, indicating that P. putida can detoxify Cd at these concentrations. EXAFS analysis of Cd bound to biomass from the different growth stages indicates that Cd binds to both sulfhydryl and non-sulfhydryl sites, but that the importance of Cd-sulfhydryl binding increases from early exponential to stationary phase. Cell growth is positively correlated to the measured sulfhydryl concentration on different biomass samples, but is independent of the measured non-sulfhydryl binding site concentration on the cell surfaces. Taken together, our results demonstrate that the sulfhydryl binding sites on EPS molecules can play an important role in binding and detoxifying toxic metals, significantly decreasing the bioavailability of the metal by sequestering it away from the bacterial cells.

Bio-organic stabilizing agent shows promising prospect for the stabilization of cadmium in contaminated farmland soil.

In situ immobilization of cadmium (Cd) has been considered as a cost-effective and non-disruptive remediation technique for Cd-contaminated soils. In this study, several immobilization approaches were compared in a Cd-contaminated agricultural farmland. The soil was treated with different combinations of the immobilizing agents such as biochar (C), rice straw (RS), lime (L), and engineered bacteria P. putida X4/pIME (B). The plant yield and Cd uptake of lettuce as well as soil Cd fractionations were measured. The Cd content in lettuce leaves and roots decreased by 46.8~67.2% and 36.8~60.2%, respectively. Among the five treatments, combined rice straw, lime, and engineered bacteria treatment showed the lowest Cd concentration in lettuce leaves (0.14 mg/kg) and the highest plant yield (21.5 t/ha). The alleviating effects are assigned to the significant transformation of water soluble and exchangeable Cd to humic substance bound, strong organic bound and residual Cd in the soils. This study suggests that this bio-organic stabilizing agent is more cost-effective than some other immobilization agents reported previously, and shows a great application prospect in improving agriculture production of heavy metal-polluted agricultural soils.

A multicenter,randomised,controlled clinical study on efficacy and safety of recombinant human interferon α 2b spray (P.putida) in treatment of acute upper respiratory tract infection in children / 中国实用儿科杂志

OBJECTIVE: To evaluate the efficacy,safety and tolerability of recombinanat human interferon α2 b(rhIFNα2b)spray in treatment of acute upper respiratory tract infection(except the flu)in children. METHODS: In total,575 children who met the inclusion criteria were enrolled from January,2019 to July,2019. They were divided into rhIFNα2b spray group(291 cases)and ribavirin group(284 cases). The children in rhIFNα2b spray group were given the rhIFNα2b spray(P.putida),and those in ribavirin groups were treated with the ribavirin spray. The curative effect and safety between the two groups were compared. RESULTS: The per-protocol set(PPS) comprised 448 patients(233 in the rhIFNα2b spray group,215 in the ribavirin group). The primary efficacy endpoint was antipyretic time which in rhIFNα2b spray group(25.0 h)was significantly shorter than that in the ribavirin group(33.6 h)(P=0.0001). In the comparison of symptomatic relief time,the relief time of nasal congestion,runny nose,sore throat and cough in rhIFNα2b spray group was shorter than that in ribavirin group(P<0.05). The recovery time in the rhIFNα2b spray group was 92 h,which showed significant decrease compared with the ribavirin group(112 h)(P<0.0001). The incidence of adverse events had no statistical differences between the two groups(P=0.2461). CONCLUSION: The rhIFNα2b spray treatment for acute upper respiratory tract infection in children is proved to be significantly effective;particularly,it can evidently relieve fever symptoms and promote the disappearance of nasal and pharyngeal symptoms. It has good safety and tolerability,so the rhIFNα2b spray is worthy to be promoted in clinical application.

bifA Regulates Biofilm Development of Pseudomonas putida MnB1 as a Primary Response to H2O2 and Mn2.

Pseudomonas putida (P. putida) MnB1 is a widely used model strain in environment science and technology for determining microbial manganese oxidation. Numerous studies have demonstrated that the growth and metabolism of P. putida MnB1 are influenced by various environmental factors. In this study, we investigated the effects of hydrogen peroxide (H2O2) and manganese (Mn2+) on proliferation, Mn2+ acquisition, anti-oxidative system, and biofilm formation of P. putida MnB1. The related orthologs of 4 genes, mco, mntABC, sod, and bifA, were amplified from P. putida GB1 and their involvement were assayed, respectively. We found that P. putida MnB1 degraded H2O2, and quickly recovered for proliferation, but its intracellular oxidative stress state was maintained, with rapid biofilm formation after H2O2 depletion. The data from mco, mntABC, sod and bifA expression levels by qRT-PCR, elucidated a sensitivity toward bifA-mediated biofilm formation, in contrary to intracellular anti-oxidative system under H2O2 exposure. Meanwhile, Mn2+ ion supply inhibited biofilm formation of P. putida MnB1. The expression pattern of these genes showed that Mn2+ ion supply likely functioned to modulate biofilm formation rather than only acting as nutrient substrate for P. putida MnB1. Furthermore, blockade of BifA activity by GTP increased the formation and development of biofilms during H2O2 exposure, while converse response to Mn2+ ion supply was evident. These distinct cellular responses to H2O2 and Mn2+ provide insights on the common mechanism by which environmental microorganisms may be protected from exogenous factors. We postulate that BifA-mediated biofilm formation but not intracellular anti-oxidative system may be a primary protective strategy adopted by P. putida MnB1. These findings will highlight the understanding of microbial adaptation mechanisms to distinct environmental stresses.

Optimal bioprocess design through a gene regulatory network - Growth kinetic hybrid model: Towards replacing Monod kinetics.

Currently, design and optimisation of biotechnological bioprocesses is performed either through exhaustive experimentation and/or with the use of empirical, unstructured growth kinetics models. Whereas, elaborate systems biology approaches have been recently explored, mixed-substrate utilisation is predominantly ignored despite its significance in enhancing bioprocess performance. Herein, bioprocess optimisation for an industrially-relevant bioremediation process involving a mixture of highly toxic substrates, m-xylene and toluene, was achieved through application of a novel experimental-modelling gene regulatory network - growth kinetic (GRN-GK) hybrid framework. The GRN model described the TOL and ortho-cleavage pathways in Pseudomonas putida mt-2 and captured the transcriptional kinetics expression patterns of the promoters. The GRN model informed the formulation of the growth kinetics model replacing the empirical and unstructured Monod kinetics. The GRN-GK framework's predictive capability and potential as a systematic optimal bioprocess design tool, was demonstrated by effectively predicting bioprocess performance, which was in agreement with experimental values, when compared to four commonly used models that deviated significantly from the experimental values. Significantly, a fed-batch biodegradation process was designed and optimised through the model-based control of TOL Pr promoter expression resulting in 61% and 60% enhanced pollutant removal and biomass formation, respectively, compared to the batch process. This provides strong evidence of model-based bioprocess optimisation at the gene level, rendering the GRN-GK framework as a novel and applicable approach to optimal bioprocess design. Finally, model analysis using global sensitivity analysis (GSA) suggests an alternative, systematic approach for model-driven strain modification for synthetic biology and metabolic engineering applications.

Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani.

Fluorescent pseudomonads from bean root and rhizosphere in Iran were investigated for biocontrol of the fungal pathogen Rhizoctonia solani. Phylogenetic analysis of concatenated 16S rRNA, gyrB and rpoD sequences for 33 Pseudomonas isolates showed that 15 belonged to four clusters within the 'P. fluorescens' group, i.e. one corresponding to P. thivervalensis, two others including P. moraviensis or P. baetica, and the last one without closely-related established species. The 18 other isolates belonged to five clusters within the 'P. putida' group, one including P. mosselii and P. entomophila, another including strains currently described as P. putida, and three without closely-related species described. Ten isolates were selected based on in vitro inhibition of R. solani. Cellulase activity was identified in three pseudomonads, chitinase activity in two pseudomonads, extracellular protease activity in nine pseudomonads and hydrogen cyanide production in two pseudomonads. Genes coding for production of phenazine, pyoluteorin, pyrrolnitrin and 2,4-diacetylphloroglucinol were not found, whereas the 1-aminocyclopropane-1-carboxylate deamination gene acdS was present in three pseudomonads. The antagonistic acdS+ strain VKh13 from the 'P. putida' group effectively protected soil-grown bean from R. solani AG 4-HGI. Results show that pseudomonads from uncharacterized taxa were readily obtained from Iranian soils and displayed biocontrol potential against R. solani.

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

INTRODUCTION: An 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. OBJECTIVE: Investigate 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: The 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.

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 Mg(2+), Ca(2+) 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 DOT­T1E strains.

Methylmercury degradation by Pseudomonas putida V1.

Environmental contamination of mercury (Hg) has caused public health concerns with focuses on the neurotoxic substance methylmercury, due to its bioaccumulation and biomagnification in food chains. The goals of the present study were to examine: (i) the transformation of methylmercury, thimerosal, phenylmercuric acetate and mercuric chloride by cultures of Pseudomonas putida V1, (ii) the presence of the genes merA and merB in P. putida V1, and (iii) the degradation pathways of methylmercury by P. putida V1. Strain V1 cultures readily degraded methylmercury, thimerosal, phenylmercury acetate, and reduced mercuric chloride into gaseous Hg(0). However, the Hg transformation in LB broth by P. putida V1 was influenced by the type of Hg compounds. The merA gene was detected in P. putida V1, on the other hand, the merB gene was not detected. The sequencing of this gene, showed high similarity (100%) to the mercuric reductase gene of other Pseudomonas spp. Furthermore, tests using radioactive (14)C-methylmercury indicated an uncommon release of (14)CO2 concomitant with the production of Hg(0). The results of the present work suggest that P. putida V1 has the potential to remove methylmercury from contaminated sites. More studies are warranted to determine the mechanism of removal of methylmercury by P. putida V1.