Pressure-dependent growth controls 3D architecture of Pseudomonas putida microcolonies.

Colony formation is key to many ecological and biotechnological processes. In its early stages, colony formation involves the concourse of a number of physical and biological parameters for generation of a distinct 3D structure-the specific influence of which remains unclear. We focused on a thus far neglected aspect of the process, specifically the consequences of the differential pressure experienced by cells in the middle of a colony versus that endured by bacteria located in the growing periphery. This feature was characterized experimentally in the soil bacterium Pseudomonas putida. Using an agent-based model we recreated the growth of microcolonies in a scenario in which pressure was the only parameter affecting proliferation of cells. Simulations exposed that, due to constant collisions with other growing bacteria, cells have virtually no free space to move sideways, thereby delaying growth and boosting chances of overlapping on top of each other. This scenario was tested experimentally on agar surfaces. Comparison between experiments and simulations suggested that the inside/outside differential pressure determines growth, both timewise and in terms of spatial directions, eventually moulding colony shape. We thus argue that-at least in the case studied-mere physical pressure of growing cells suffices to explain key dynamics of colony formation.

Enhancement of drought tolerance in diverse Vicia faba cultivars by inoculation with plant growth-promoting rhizobacteria under newly reclaimed soil conditions.

Water deficit has devastating impacts on legume production, particularly with the current abrupt climate changes in arid environments. The application of plant growth-promoting rhizobacteria (PGPR) is an effective approach for producing natural nitrogen and attenuating the detrimental effects of drought stress. This study investigated the influence of inoculation with the PGPR Rhizobium leguminosarum biovar viciae (USDA 2435) and Pseudomonas putida (RA MTCC5279) solely or in combination on the physio-biochemical and agronomic traits of five diverse Vicia faba cultivars under well-watered (100% crop evapotranspiration [ETc]), moderate drought (75% ETc), and severe drought (50% ETc) conditions in newly reclaimed poor-fertility sandy soil. Drought stress substantially reduced the expression of photosynthetic pigments and water relation parameters. In contrast, antioxidant enzyme activities and osmoprotectants were considerably increased in plants under drought stress compared with those in well-watered plants. These adverse effects of drought stress reduced crop water productivity (CWP) and seed yield-related traits. However, the application of PGPR, particularly a consortium of both strains, improved these parameters and increased seed yield and CWP. The evaluated cultivars displayed varied tolerance to drought stress: Giza-843 and Giza-716 had the highest tolerance under well-watered and moderate drought conditions, whereas Giza-843 and Sakha-4 were more tolerant under severe drought conditions. Thus, co-inoculation of drought-tolerant cultivars with R. leguminosarum and P. putida enhanced their tolerance and increased their yield and CWP under water-deficit stress conditions. This study showed for the first time that the combined use of R. leguminosarum and P. putida is a promising and ecofriendly strategy for increasing drought tolerance in legume crops.

High-throughput screening alternative to crystal violet biofilm assay combining fluorescence quantification and imaging.

The crystal violet assay is widely used for biofilm quantitation despite its toxicity and variability. Here, we instead combine fluorescence labelling with the Cytation 5 multi-mode plate reader, to enable simultaneous acquisition of both quantitative and imaging biofilm data. This high-throughput method produces more robust data and provides information about morphology and spatial species organization within the biofilm.

Transformation of Iodosulfuron-Methyl into Ionic Liquids Enables Elimination of Additional Surfactants in Commercial Formulations of Sulfonylureas.

Efficient use of herbicides for plant protection requires the application of auxiliary substances such as surfactants, stabilizers, wetting or anti-foaming agents, and absorption enhancers, which can be more problematic for environment than the herbicides themselves. We hypothesized that the combination of sulfonylurea (iodosulfuron-methyl) anion with inexpensive, commercially available quaternary tetraalkylammonium cations could lead to biologically active ionic liquids (ILs) that could become a convenient and environment-friendly alternative to adjuvants. A simple one-step synthesis allowed for synthesizing iodosulfuron-methyl based ILs with high yields ranging from 88 to 96% as confirmed by UV, FTIR, and NMR. The obtained ILs were found to possess several favorable properties compared to the currently used sodium salt iodosulfuron-methyl, such as adjustable hydrophobicity (octanol-water partition coefficient) and enhanced stability in aqueous solutions, which was supported by molecular calculations showing cation-anion interaction energies. In addition, soil mobility and volatility of ILs were more beneficial compared to the parental herbicide. Herbicidal activity tests toward oil-seed rape and cornflower revealed that ILs comprising at least one alkyl chain in the decyl to octadecyl range had similar or better efficacy compared to the commercial preparation without addition of any adjuvant. Furthermore, results of antimicrobial activity indicated that they were practically harmless or slightly toxic toward model soil microorganisms such as Pseudomonas putida and Bacillus cereus.

An Exploration of Seaweed Polysaccharides Stimulating Denitrifying Bacteria for Safer Nitrate Removal.

Excessive use of nitrogen fertilizer in intensively managed agriculture has resulted in abundant accumulation of nitrate in soil, which limits agriculture sustainability. How to reduce nitrate content is the key to alleviate secondary soil salinization. However, the microorganisms used in soil remediation cause some problems such as weak efficiency and short survival time. In this study, seaweed polysaccharides were used as stimulant to promote the rapid growth and safer nitrate removal of denitrifying bacteria. Firstly, the growth rate and NO3--N removal capacity of three kinds of denitrifying bacteria, Bacillus subtilis (BS), Pseudomonas stutzeri (PS) and Pseudomonas putida (PP), were compared. The results showed that Bacillus subtilis (BS) had a faster growth rate and stronger nitrate removal ability. We then studied the effects of Enteromorpha linza polysaccharides (EP), carrageenan (CA), and sodium alginate (AL) on growth and denitrification performance of Bacillus subtilis (BS). The results showed that seaweed polysaccharides obviously promoted the growth of Bacillus subtilis (BS), and accelerated the reduction of NO3--N. More importantly, the increased NH4+-N content could avoid excessive loss of nitrogen, and less NO2--N accumulation could avoid toxic effects on plants. This new strategy of using denitrifying bacteria for safely remediating secondary soil salinization has a great significance.

Aedes aegypti Toll pathway is induced through dsRNA sensing in endosomes.

Mosquito anti-pathogen immune responses, including those controlling infection with arboviruses are regulated by multiple signal transduction pathways. While the Toll pathway is critical in the defense against arboviruses such as dengue and Zika viruses, the factors and mechanisms involved in virus recognition leading to the activation of the Toll pathway are not fully understood. In this study we evaluated the role of virus-produced double-stranded RNA (dsRNA) intermediates in mosquito immune activation by utilizing the synthetic dsRNA analog polyinosinic-polycytidylic acid (poly I:C). Poly I:C treatment of Aedes aegypti mosquitoes and Aag2 cells reduced DENV infection. Transcriptomic analyses of Aag2 cell responses to poly I:C indicated putative activation of the Toll pathway. We found that poly I:C is translocated to the endosomal compartment of Aag2 cells, and that the A. aegypti Toll 6 receptor is a putative dsRNA recognition receptor. This study elucidates the role of dsRNAs in the immune activation of non-RNAi pathways in mosquitoes.

Productivity of Pseudomonas putida TISTR 1522 in polyhydroxyalkanoates (PHAs) production from saponified palm oil.

Polyhydroxyalkanoates (PHAs) have attracted attention as an environmentally degradable bioplastic which potentially replaces synthetic polymers used in a wide range of industries. One of most promising microorganisms for the production of PHAs is Pseudomonas putida. In this study, we purpose to develop sustainable processes to convert abundant palm oil available in local market to high value PHAs and optimize PHAs production by Pseudomonas putida TISTR 1522 from saponified palm oil. We found that the highest yield of PHAs production (0.95 g/L, 40.15%) was obtained in culture medium supplemented with 1% (w/v) fatty acid salt by P. putida TISTR 1522 after 24-h cultivation. The intracellular PHAs were located in granules inside the cells, which fluoresced bright yellow by staining with Nile red. The physical appearance of intracellular PHAs investigated by transmission electron microscope (TEM) revealed that PHAs accumulate in granules, about 3-10 granules per cell. These granules are white and roundish-shaped with 0.3-0.5-µm diameter. The 1H NMR spectrum represented the typical characters of medium-chain length-PHAs. This variation of all parameters was successfully demonstrated a good intracellular PHAs accumulation in P. putida TISTR 1522 by fatty acid salt utilization.

A metabolic and physiological design study of Pseudomonas putida KT2440 capable of anaerobic respiration.

BACKGROUND: Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and thus interesting microbial chassis for biotechnological applications. However, its obligate aerobic nature hampers production of oxygen sensitive products and drives up costs in large scale fermentation. The inability to perform anaerobic fermentation has been attributed to insufficient ATP production and an inability to produce pyrimidines under these conditions. Addressing these bottlenecks enabled growth under micro-oxic conditions but does not lead to growth or survival under anoxic conditions. RESULTS: Here, a data-driven approach was used to develop a rational design for a P. putida KT2440 derivative strain capable of anaerobic respiration. To come to the design, data derived from a genome comparison of 1628 Pseudomonas strains was combined with genome-scale metabolic modelling simulations and a transcriptome dataset of 47 samples representing 14 environmental conditions from the facultative anaerobe Pseudomonas aeruginosa. CONCLUSIONS: The results indicate that the implementation of anaerobic respiration in P. putida KT2440 would require at least 49 additional genes of known function, at least 8 genes encoding proteins of unknown function, and 3 externally added vitamins.

Temperature regulation as a tool to program synthetic microbial community composition.

Engineering of synthetic microbial communities is emerging as a powerful new paradigm for performing various industrially, medically, and environmentally important processes. To reach the fullest potential, however, this approach requires further development in many aspects, a key one being regulating the community composition. Here we leverage well-established mechanisms in ecology which govern the relative abundance of multispecies ecosystems and develop a new tool for programming the composition of synthetic microbial communities. Using a simple model system consisting of two microorganisms Escherichia coli and Pseudomonas putida, which occupy different but partially overlapping thermal niches, we demonstrated that temperature regulation could be used to enable coexistence and program the community composition. We first investigated a constant temperature regime and showed that different temperatures led to different community compositions. Next, we invented a new cycling temperature regime and showed that it can dynamically tune the microbial community, achieving a wide range of compositions depending on parameters that are readily manipulatable. Our work provides conclusive proof of concept that temperature regulation is a versatile and powerful tool capable of programming compositions of synthetic microbial communities.

Isolation, identification and characterization of phenolic acid-degrading bacteria from soil.

AIMS: To isolate, identify and characterize phenolic acid-degrading bacteria and reduce plant growth inhibition caused by phenolic acids. METHODS AND RESULTS: A total of 11 bacterial isolates with high phthalic acid (PA)-degrading ability were obtained using mineral salt medium (MSM) medium containing PA as sole carbon source. These isolates were identified as Arthrobacter globiformis, Pseudomonas putida and Pseudomonas hunanensis by sequence analyses of the 16S rRNA gene. Among them, five Pseudomonas strains could also effectively degrade ferulic acid (FA), p-hydroxybenzoic acid (PHBA) and syringic acid (SA) in MSM solution. P. putida strain 7 and P. hunanensis strain 10 showed highly efficient degradation of PA, SA, FA and PHBA, and could reduce their inhibition of lily, watermelon, poplar and strawberry seedling growth in soils respectively. These two strains could promote plant growth in soil with phenolic acids. CONCLUSIONS: In this study, bacterial strains with highly efficient phenolic acid-degrading abilities could not only effectively reduce the autotoxicity of phenolic acids on plants but also were able to promote plant growth in soil with phenolic acids. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, Pseudomonas can promote plant growth while degrading phenolic acids. Our results provide new choices for the biological removal of autotoxins.

Bacterial inoculation positively affects the quality and quantity of flax under deficit irrigation regimes.

AIM: The present research was conducted to investigate the effect of plant growth-promoting rhizobacteria (PGPR) and deficit irrigation on quality and quantity of flax under field and pot conditions to determine bacterial efficiency and to decrease water deficit effects. METHODS AND RESULTS: Initially, in vitro experiments were performed to determine the growth-promoting characteristics of bacteria. Then in the field, the effects of bacterial inoculation (control, Azotobacter chroococcum, Azospirillum lipoferum, Bacillus amyloliquefaciens, Bacillus sp. strain1 and Pseudomonas putida) on flax traits were evaluated at different irrigation levels (100, 75 and 50% crop water requirement). Bacterial treatments in the pot experiment were selected based on the field experiment results. The irrigation regimes in the pot and field experiments were the same and bacterial treatments included single, doublet and triplet applications of the bacteria. All the bacterial strains could solubilize phosphate, produce ammonia (except for Bacillus sp. strain1), indole acetic acid and siderophore (except P. putida). Field results indicated that the bacteria significantly mitigated the effects of water deficit. Compared with control plants, bacterial treatments increased the oil, linolenic acid, protein and sulphur content; the number of shoots and capsules; and the harvest index in the flax plants. Pot experimental results revealed that the combined inoculations were more effective than single inoculum treatments. CONCLUSIONS: Bacterial inoculation alleviates deficit irrigation effects in flax plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The effectiveness of applying A. chroococcum, B. amyloliquefaciens and Bacillus sp. strain1 was confirmed, especially as a combination to protect flax against water deficit and to improve its nutritional quality and growth.

The putative phosphate transporter PitB (PP1373) is involved in tellurite uptake in Pseudomonas putida KT2440.

Tellurium oxyanions are chemical species of great toxicity and their presence in the environment has increased because of mining industries and photovoltaic and electronic waste. Recovery strategies for this metalloid that are based on micro-organisms are of interest, but further studies of the transport systems and enzymes responsible for implementing tellurium transformations are required because many mechanisms remain unknown. Here, we investigated the involvement in tellurite uptake of the putative phosphate transporter PitB (PP1373) in soil bacterium Pseudomonas putida KT2440. For this purpose, through a method based on the CRISPR/Cas9 system, we generated a strain deficient in the pitB gene and characterized its phenotype on exposing it to varied concentrations of tellurite. Growth curves and transmission electronic microscopy experiments for the wild-type and ΔpitB strains showed that both were able to internalize tellurite into the cytoplasm and reduce the oxyanion to black nano-sized and rod-shaped tellurium particles, although the ΔpitB strain showed an increased resistance to the tellurite toxic effects. At a concentration of 100 µM tellurite, where the biomass formation of the wild-type strain decreased by half, we observed a greater ability of ΔpitB to reduce this oxyanion with respect to the wild-type strain (~38 vs ~16 %), which is related to the greater biomass production of ΔpitB and not to a greater consumption of tellurite per cell. The phenotype of the mutant was restored on over-expressing pitB in trans. In summary, our results indicate that PitB is one of several transporters responsible for tellurite uptake in P. putida KT2440.

Carbon Source Influence on Extracellular pH Changes along Bacterial Cell-Growth.

The effect of initial pH on bacterial cell-growth and its change over time was studied under aerobic heterotrophic conditions by using three bacterial strains: Escherichia coli ATCC 25922, Pseudomonas putida KT2440, and Pseudomonas pseudoalcaligenes CECT 5344. In Luria-Bertani (LB) media, pH evolved by converging to a certain value that is specific for each bacterium. By contrast, in the buffered Minimal Medium (MM), pH was generally more stable along the growth curve. In MM with glucose as carbon source, a slight acidification of the medium was observed for all strains. In the case of E. coli, a sudden drop in pH was observed during exponential cell growth that was later recovered at initial pH 7 or 8, but was irreversible below pH 6, thus arresting further cell-growth. When using other carbon sources in MM at a fixed initial pH, pH changes depended mainly on the carbon source itself. While glucose, glycerol, or octanoate slightly decreased extracellular pH, more oxidized carbon sources, such as citrate, 2-furoate, 2-oxoglutarate, and fumarate, ended up with the alkalinization of the medium. These observations are in accordance with pH change predictions using genome-scale metabolic models for the three strains, thus revealing the metabolic reasons behind pH change. Therefore, we conclude that the composition of the medium, specifically the carbon source, determines pH change during bacterial growth to a great extent and unravel the main molecular mechanism behind this phenotype. These findings pave the way for predicting pH changes in a given bacterial culture and may anticipate the interspecies interactions and fitness of bacteria in their environment.

Adaptive laboratory evolution of Pseudomonas putida and Corynebacterium glutamicum to enhance anthranilate tolerance.

Microbial bioproduction of the aromatic acid anthranilate (ortho-aminobenzoate) has the potential to replace its current, environmentally demanding production process. The host organism employed for such a process needs to fulfil certain demands to achieve industrially relevant product levels. As anthranilate is toxic for microorganisms, the use of particularly robust production hosts can overcome issues from product inhibition. The microorganisms Corynebacterium glutamicum and Pseudomonas putida are known for high tolerance towards a variety of chemicals and could serve as promising platform strains. In this study, the resistance of both wild-type strains towards anthranilate was assessed. To further enhance their native tolerance, adaptive laboratory evolution (ALE) was applied. Sequential batch fermentation processes were developed, adapted to the cultivation demands for C. glutamicum and P. putida, to enable long-term cultivation in the presence of anthranilate. Isolation and analysis of single mutants revealed phenotypes with improved growth behaviour in the presence of anthranilate for both strains. The characterization and improvement of both potential hosts provide an important basis for further process optimization and will aid the establishment of an industrially competitive method for microbial synthesis of anthranilate.

Arginine as an environmental and metabolic cue for cyclic diguanylate signalling and biofilm formation in Pseudomonas putida.

Cyclic diguanylate (c-di-GMP) is a broadly conserved intracellular second messenger that influences different bacterial processes, including virulence, stress tolerance or social behaviours and biofilm development. Although in most cases the environmental cue that initiates the signal transduction cascade leading to changes in cellular c-di-GMP levels remains unknown, certain L- and D-amino acids have been described to modulate c-di-GMP turnover in some bacteria. In this work, we have analysed the influence of L-amino acids on c-di-GMP levels in the plant-beneficial bacterium Pseudomonas putida KT2440, identifying L-arginine as the main one causing a significant increase in c-di-GMP. Both exogenous (environmental) and endogenous (biosynthetic) L-arginine influence biofilm formation by P. putida through changes in c-di-GMP content and altered expression of structural elements of the biofilm extracellular matrix. The contribution of periplasmic binding proteins forming part of amino acid transport systems to the response to environmental L-arginine was also studied. Contrary to what has been described in other bacteria, in P. putida these proteins seem not to be directly responsible for signal transduction. Rather, their contribution to global L-arginine pools appears to determine changes in c-di-GMP turnover. We propose that arginine plays a connecting role between cellular metabolism and c-di-GMP signalling in P. putida.

Exploiting geometric similarity for statistical quantification of fluorescence spatial patterns in bacterial colonies.

BACKGROUND: Currently the combination of molecular tools, imaging techniques and analysis software offer the possibility of studying gene activity through the use of fluorescent reporters and infer its distribution within complex biological three-dimensional structures. For example, the use of Confocal Scanning Laser Microscopy (CSLM) is a regularly-used approach to visually inspect the spatial distribution of a fluorescent signal. Although a plethora of generalist imaging software is available to analyze experimental pictures, the development of tailor-made software for every specific problem is still the most straightforward approach to perform the best possible image analysis. In this manuscript, we focused on developing a simple methodology to satisfy one particular need: automated processing and analysis of CSLM image stacks to generate 3D fluorescence profiles showing the average distribution detected in bacterial colonies grown in different experimental conditions for comparison purposes. RESULTS: The presented method processes batches of CSLM stacks containing three-dimensional images of an arbitrary number of colonies. Quasi-circular colonies are identified, filtered and projected onto a normalized orthogonal coordinate system, where a numerical interpolation is performed to obtain fluorescence values within a spatially fixed grid. A statistically representative three-dimensional fluorescent pattern is then generated from this data, allowing for standardized fluorescence analysis regardless of variability in colony size. The proposed methodology was evaluated by analyzing fluorescence from GFP expression subject to regulation by a stress-inducible promoter. CONCLUSIONS: This method provides a statistically reliable spatial distribution profile of fluorescence detected in analyzed samples, helping the researcher to establish general correlations between gene expression and spatial allocation under differential experimental regimes. The described methodology was coded into a MATLAB script and shared under an open source license to make it accessible to the whole community.

The versatility of Pseudomonas putida in the rhizosphere environment.

This article addresses the lifestyle of Pseudomonas and focuses on how Pseudomonas putida can be used as a model system for biotechnological processes in agriculture, and in the removal of pollutants from soils. In this chapter we aim to show how a deep analysis using genetic information and experimental tests has helped to reveal insights into the lifestyle of Pseudomonads. Pseudomonas putida is a Plant Growth Promoting Rhizobacteria (PGPR) that establishes commensal relationships with plants. The interaction involves a series of functions encoded by core genes which favor nutrient mobilization, prevention of pathogen development and efficient niche colonization. Certain Pseudomonas putida strains harbor accessory genes that confer specific biodegradative properties and because these microorganisms can thrive on the roots of plants they can be exploited to remove pollutants via rhizoremediation, making the consortium plant/Pseudomonas a useful tool to combat pollution.

Parameter identification for gompertz and logistic dynamic equations.

In this paper, we generalize and compare Gompertz and Logistic dynamic equations in order to describe the growth patterns of bacteria and tumor. First of all, we introduce two types of Gompertz equations, where the first type 4-paramater and 3-parameter Gompertz curves do not include the logarithm of the number of individuals, and then we derive 4-parameter and 3-parameter Logistic equations. We notice that Logistic curves are better in modeling bacteria whereas the growth pattern of tumor is described better by Gompertz curves. Increasing the number of parameters of Logistic curves give favorable results for bacteria while decreasing the number of parameters of Gompertz curves for tumor improves the curve fitting. Moreover, our results overshadow some of the existing results in the literature.

Biotransformation of 5-Hydroxymethylfurfural to 2,5-Furandicarboxylic Acid by a Syntrophic Consortium of Engineered Synechococcus elongatus and Pseudomonas putida.

2,5-furandicarboxylic acid (FDCA) is one of the top platform chemicals that can be produced from biomass feedstock. To make the cost of industrial FDCA production compatible with plastics made from fossils, the price of substrates and process complexity should be reduced. The aim of this research is to create a CO2 -driven syntrophic consortium for the catalytic conversion of renewable biomass-derived 5-hydroxymethylfurfural (HMF) to FDCA. Sucrose produced from carbon fixation by the engineered Synechococcus elongatus serves as the sole carbon source for the engineered Pseudomonas putida to catalyze the reaction of HMF to FDCA. The yield of FDCA by the consortium reaches around 70% while the conversion of HMF is close to 100%. With further surface engineering to clump the two strains, the FDCA yield is elevated to almost 100% via the specific association between an Src homology 3 (SH3) domain and its ligand. The syntrophic consortium successfully demonstrates its green and cost-effective characteristics for the conversion of CO2 and biomass into platform chemicals.

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.