Reconstitution of a biofilm adhesin system from a sulfate-reducing bacterium in Pseudomonas fluorescens.

Biofilms of sulfate-reducing bacterium (SRB) like Desulfovibrio vulgaris Hildenborough (DvH) can facilitate metal corrosion in various industrial and environmental settings leading to substantial economic losses. Although the mechanisms of biofilm formation by DvH are not yet well understood, recent studies indicate the large adhesin, DvhA, is a key determinant of biofilm formation. The dvhA gene neighborhood resembles the biofilm-regulating Lap system of Pseudomonas fluorescens but is curiously missing the c-di-GMP-binding regulator LapD. Instead, DvH encodes an evolutionarily unrelated c-di-GMP-binding protein (DVU1020) that we hypothesized is functionally analogous to LapD. To study this unusual Lap system and overcome experimental limitations with the slow-growing anaerobe DvH, we reconstituted its predicted SRB Lap system in a P. fluorescens strain lacking its native Lap regulatory components (ΔlapGΔlapD). Our data support the model that DvhA is a cell surface-associated LapA-like adhesin with a N-terminal "retention module" and that DvhA is released from the cell surface upon cleavage by the LapG-like protease DvhG. Further, we demonstrate DVU1020 (named here DvhD) represents a distinct class of c-di-GMP-binding, biofilm-regulating proteins that regulates DvhG activity in response to intracellular levels of this second messenger. This study provides insight into the key players responsible for biofilm formation by DvH, thereby expanding our understanding of Lap-like systems.

Chemotaxis-mediated degradation of PAHs and heterocyclic PAHs under low-temperature stress by Pseudomonas fluorescens S01: Insights into the mechanisms of biodegradation and cold adaptation.

As wellknown persistent contaminants, polycyclic aromatic hydrocarbons (PAHs) and heterocyclic polyaromatic hydrocarbons (Heterocyclic PAHs)'s fates in cryogenic environments are remains uncertain. Herein, strain S01 was identified as Pseudomonas fluorescens, a novel bacterium tolerant to low temperature and capable of degrading PAHs and heterocyclic PAHs. Strain S01 exhibited growth at 5-40 â„ƒ and degradation rate of mixed PAHs and heterocyclic PAHs reached 52% under low-temperature. Through comprehensive metabolomic, genomic, and transcriptomic analyses, we reconstructed the biodegradation pathway for PAHs and heterocyclic PAHs in S01 while investigating its response to low temperature. Further experiments involving deletion and replacement of methyl-accepting chemotaxis protein (MCP) confirmed its crucial role in enabling strain S01's adaptation to dual stress of low temperature and pollutants. Additionally, our analysis revealed that MCP was upregulated under cold stress which enhanced strain S01's motility capabilities leading to increased biofilm formation. The establishment of biofilm promoted preservation of distinct cellular membrane stability, thereby enhancing energy metabolism. Consequently, this led to heightened efficiency in pollutant degradation and improved cold resistance capabilities. Our findings provide a comprehensive understanding of the environmental fate of both PAHs and heterocyclic PAHs under low-temperature conditions while also shedding light on cold adaptation mechanism employed by strain S01.

Conserved Signal Transduction Mechanisms and Dark Recovery Kinetic Tuning in the Pseudomonadaceae Short Light, Oxygen, Voltage (LOV) Protein Family.

Light-Oxygen-Voltage (LOV) flavoproteins transduce a light signal into variable signaling outputs via a structural rearrangement in the sensory core domain, which is then relayed to fused effector domains via α-helical linker elements. Short LOV proteins from Pseudomonadaceae consist of a LOV sensory core and N- and C-terminal α-helices of variable length, providing a simple model system to study the molecular mechanism of allosteric activation. Here we report the crystal structures of two LOV proteins from Pseudomonas fluorescens - SBW25-LOV in the fully light-adapted state and Pf5-LOV in the dark-state. In a comparative analysis of the Pseudomonadaceae short LOVs, the structures demonstrate light-induced rotation of the core domains and splaying of the proximal A'α and Jα helices in the N and C-termini, highlighting evidence for a conserved signal transduction mechanism. Another distinguishing feature of the Pseudomonadaceae short LOV protein family is their highly variable dark recovery, ranging from seconds to days. Understanding this variability is crucial for tuning the signaling behavior of LOV-based optogenetic tools. At 37 °C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. To investigate this remarkable difference in dark recovery rates, we targeted three residues lining the solvent channel entrance to the chromophore pocket where we introduced mutations by exchanging the non-conserved amino acids from SBW25-LOV into Pf5-LOV and vice versa. Dark recovery kinetics of the resulting mutants, as well as MD simulations and solvent cavity calculations on the crystal structures suggest a correlation between solvent accessibility and adduct lifetime.

Synergistic action of Pseudomonas fluorescens with melatonin attenuates salt toxicity in mustard by regulating antioxidant system and flavonoid profile.

Salt stress is an alarming abiotic stress that reduces mustard growth and yield. To attenuate salt toxicity effects, plant growth-promoting rhizobacteria (PGPR) offers a sustainable approach. Among the various PGPR, Pseudomonas fluorescens (P. fluorescens NAIMCC-B-00340) was chosen for its salt tolerance (at 100 mM NaCl) and for exhibiting various growth-promoting activities. Notably, P. fluorescens can produce auxin, which plays a role in melatonin (MT) synthesis. Melatonin is a pleiotropic molecule that acts as an antioxidant to scavenge reactive oxygen species (ROS), resulting in stress reduction. Owing to the individual role of PGPR and MT in salt tolerance, and their casual nexus, their domino effect was investigated in Indian mustard under salt stress. The synergistic action of P. fluorescens and MT under salt stress conditions was found to enhance the activity of antioxidative enzymes and proline content as well as  promote the production of secondary metabolites. This led to reduced oxidative stress following effective ROS scavenging, maintained photosynthesis, and improved growth. In mustard plants treated with MT and P. fluorescens under salt stress, eight flavonoids showed significant increase. Kaempferol and cyanidin showed the highest concentrations and are reported to act as antioxidants with protective functions under stress. Thus, we can anticipate that strategies involved in their enhancement could provide a better adaptive solution to salt toxicity in mustard plants. In conclusion, the combination of P. fluorescens and MT affected antioxidant metabolism and flavonoid profile that could be used to mitigate salt-induced stress and bolster plant resilience.

Pseudomonas fluorescens and L-tryptophan application triggered the phytoremediation potential of sunflower (Heliantus annuus L.) in lead-contaminated soil.

Lead, a toxic heavy metal present in soil, hampers biological activities and affects the metabolism of plants, animals, and human beings. Its higher concentration may disturb the various physio-chemical processes, which result in stunted and poor plant growth. An interactive approach of plant growth promoting rhizobacteria (PGPR) and L-tryptophan can be used to mitigate the lethal effects of lead. A pot experiment was conducted, and two weeks before sowing, the level of lead (300 mg kg-1) was maintained by spiking the PbCl2 salt. Pseudomonas fluorescens and L-tryptophan were applied individually as well as in combination to segregate the effect of both in contaminated soil under a completely Randomized Design (CRD). Statistical analysis revealed that plant growth was significantly reduced up to 22% due to lead contamination. However, the interactive approach of PGPR and L-tryptophan significantly improved the plant growth, physiology, and yield with relative productive index (RPI) under a lead-stressed environment. Moreover, integrated use of PGPR and L-tryptophan demonstrated a considerable increase (22%) in lead removal efficiency (LRE) by improving bioconcentration factor (BCF) and translocation factor (TF) for shoot without increasing the lead concentration in achenes. The reduced lead concentration in achene was due to its immobilization in shoot and root by negatively charged particles and improved the lead sequestration in vegetative parts which abridged the translocation of lead into achenes.

Hypertonic stress induced changes of Pseudomonas fluorescens adhesion towards soil minerals studied by AFM.

Studying bacterial adhesion to mineral surfaces is crucial for understanding soil properties. Recent research suggests that minimal coverage of sand particles with cell fragments significantly reduces soil wettability. Using atomic force microscopy (AFM), we investigated the influence of hypertonic stress on Pseudomonas fluorescens adhesion to four different minerals in water. These findings were compared with theoretical XDLVO predictions. To make adhesion force measurements comparable for irregularly shaped particles, we normalized adhesion forces by the respective cell-mineral contact area. Our study revealed an inverse relationship between wettability and the surface-organic carbon content of the minerals. This relationship was evident in the increased adhesion of cells to minerals with decreasing wettability. This phenomenon was attributed to hydrophobic interactions, which appeared to be predominant in all cell-mineral interaction scenarios alongside with hydrogen bonding. Moreover, while montmorillonite and goethite exhibited stronger adhesion to stressed cells, presumably due to enhanced hydrophobic interactions, kaolinite showed an unexpected trend of weaker adhesion to stressed cells. Surprisingly, the adhesion of quartz remained independent of cell stress level. Discrepancies between measured cell-mineral interactions and those calculated by XDLVO, assuming an idealized sphere-plane geometry, helped us interpret the chemical heterogeneity arising from differently exposed edges and planes of minerals. Our results suggest that bacteria may have a significant impact on soil wettability under changing moisture condition.

Pseudomonas fluorescens MFE01 delivers a putative type VI secretion amidase that confers biocontrol against the soft-rot pathogen Pectobacterium atrosepticum.

The type VI secretion system (T6SS) is a contractile nanomachine widespread in Gram-negative bacteria. The T6SS injects effectors into target cells including eukaryotic hosts and competitor microbial cells and thus participates in pathogenesis and intermicrobial competition. Pseudomonas fluorescens MFE01 possesses a single T6SS gene cluster that confers biocontrol properties by protecting potato tubers against the phytopathogen Pectobacterium atrosepticum (Pca). Here, we demonstrate that a functional T6SS is essential to protect potato tuber by reducing the pectobacteria population. Fluorescence microscopy experiments showed that MFE01 displays an aggressive behaviour with an offensive T6SS characterized by continuous and intense T6SS firing activity. Interestingly, we observed that T6SS firing is correlated with rounding of Pectobacterium cells, suggesting delivery of a potent cell wall targeting effector. Mutagenesis coupled with functional assays then revealed that a putative T6SS secreted amidase, Tae3Pf , is mainly responsible for MFE01 toxicity towards Pca. Further studies finally demonstrated that Tae3Pf is toxic when produced in the periplasm, and that its toxicity is counteracted by the Tai3Pf inner membrane immunity protein.

Characterization and metabolic pathway of Pseudomonas fluorescens 2P24 for highly efficient ammonium and nitrate removal.

The ammonium and nitrate removal performance and metabolic pathways of a biocontrol strain, Pseudomonas fluorescens 2P24, were investigated. Strain 2P24 could completely remove 100 mg/L ammonium and nitrate, with removal rates of 8.27 mg/L/h and 4.29 mg/L/h, respectively. During these processes, most of the ammonium and nitrate were converted to biological nitrogen via assimilation, and only small amounts of nitrous oxide escaped. The inhibitor allylthiourea had no impact on ammonium transformation, and diethyl dithiocarbamate and sodium tungstate did not inhibit nitrate removal. Intracellular nitrate and ammonium were detectable during the nitrate and ammonium transformation process, respectively. Moreover, the nitrogen metabolism functional genes (glnK, nasA, narG, nirBD, nxrAB, nirS, nirK, and norB) were identified in the strain. All results highlighted that P. fluorescens 2P24 is capable of assimilatory and dissimilatory nitrate reduction, ammonium assimilation and oxidation, and denitrification.

Characterization of a New Thermostable and Organic Solution-Tolerant Lipase from Pseudomonas fluorescens and Its Application in the Enrichment of Polyunsaturated Fatty Acids.

The search for and characterization of new lipases with excellent properties has always been urgent and is of great importance to meet industrial needs. In this study, a new lipase, lipB, from Pseudomonas fluorescens SBW25, belonging to the lipase subfamily I.3, was cloned and expressed in Bacillus subtilis WB800N. Enzymatic properties studies of recombinant LipB found that it exhibited the highest activity towards p-nitrophenyl caprylate at 40 °C and pH 8.0, retaining 73% of its original activity after incubation at 70 °C for 6 h. In addition, Ca2+, Mg2+, and Ba2+ strongly enhanced the activity of LipB, while Cu2+, Zn2+, Mn2+, and CTAB showed an inhibiting effect. The LipB also displayed noticeable tolerance to organic solvents, especially acetonitrile, isopropanol, acetone, and DMSO. Moreover, LipB was applied to the enrichment of polyunsaturated fatty acids from fish oil. After hydrolyzing for 24 h, it could increase the contents of polyunsaturated fatty acids from 43.16% to 72.18%, consisting of 5.75% eicosapentaenoic acid, 19.57% docosapentaenoic acid, and 46.86% docosahexaenoic acid, respectively. The properties of LipB render it great potential in industrial applications, especially in health food production.

Triclocarban-contaminated wastewater treatment by innovative hybrid moving entrapped bead activated sludge reactor (HyMER): Continuous performance and computational dynamic simulation analysis.

Triclocarban (TCC) has been used in consumer products and is a widespread contaminant in municipal wastewater treatment systems that ultimately accumulates in natural receiving water and soil. This work aims to apply an innovative hybrid moving entrapped bead activated sludge reactor (named "HyMER") that integrates entrapped TCC-degrading microbes and freely suspended activated sludge to treat TCC-contaminated wastewater. A previously isolated TCC-degrading bacterium (Pseudomonas fluorescens strain MC46, called MC46) and barium alginate entrapment were applied. The synthetic TCC-contaminated wastewater treatment (with TCC concentration of 10 mg/L) was performed using 20-cycle fed-batch reactor operation with feeding times of 12 and 24 h and cycle times of 13 and 25 h. The results indicated that the HyMER effectively reduced chemical oxygen demand by up to 80 and 95 % and TCC by up to 53 and 83 %, respectively, with feeding times of 12 and 24 h. Three TCC degradation intermediate products were found-3,4-dichloroaniline, 4-chloroaniline, and aniline. Scanning electron microscopic analysis revealed shorter cells and bacterial appendage development as cell adaptations against TCC and its intermediates. The live/dead assay indicated high survival of entrapped MC46 in toxic conditions, with up to 84 % viable cells. Based on computational fluid dynamic analysis, no entrapped cell agglomeration showed in the reactor, indicating the potential application of HyMER for real wastewater treatment. These results exhibit the feasibility of HyMER and its applicability for future toxic wastewater treatment.

Glutamate Positively Regulates Chitinase Activity and the Biocontrol Efficacy of Pseudomonas protegens.

Broad-spectrum biocontrol by Pseudomonas protegens CHA0 and other fluorescent pseudomonads is achieved through the generation of various secondary metabolites with antibiotic activities against not only other microbes but, also, nematodes and insects present in the rhizosphere. A previous metabolomic study demonstrated that intracellular low-molecular weight effectors, such as guanosine tetraphosphate and γ-aminobutyrate, function as important signals in niche adaptation by strain CHA0 to plant roots. We investigated the role of amino acids in the biocontrol trait of P. protegens Cab57 towards Pythium damping off and root rot in cucumber. Among the 11 amino acids tested, only glutamate markedly enhanced the efficacy of biocontrol. An RNA-Seq analysis revealed that glutamate upregulated the expression of a chitinase gene cluster (c21370-c21380, in which the c21370 gene was annotated as a gene encoding the chitin-binding protein cbp and the c21380 gene encoded chitinase chiC) in strain CHA0. Glutamate upregulated the expression of the regulatory small RNA rsmZ but reduced the production levels of other Gac/Rsm-regulated biocontrol factors, such as 2,4-diacetylphloroglucinol and pyoluteorin. The promoter activity of cbp and chitinase activity were characterized in detail; their activities were up-regulated in response to glutamate and their expression was under the control of GacA. Therefore, glutamate appears to be essential for biocontrol activity in which chitinase production is regulated in response to glutamate. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Antifungal mechanisms of volatile organic compounds produced by Pseudomonas fluorescens ZX as biological fumigants against Botrytis cinerea.

To explore the antifungal mechanisms of volatile organic compounds (VOCs) produced by Pseudomonas fluorescens ZX against Botrytis cinerea, biochemical analyses and transcriptomic techniques were employed in this work. The results showed that P. fluorescens ZX-producing VOCs can increase the cell membrane permeability of B. cinerea and disrupt cell membrane integrity, resulting in leakage of the pathogen's cellular contents, inhibition of ergosterol biosynthesis (about 76%), and an increase in malondialdehyde (MDA) content. Additionally, for B. cinerea respiration, P. fluorescens ZX-producing VOCs (1 × 109 CFU /mL) significantly inhibited the activities of ATPase (55.7%), malate dehydrogenase (MDH) (33.1%), and succinate dehydrogenase (SDH) (57.9%), seriously interfering with energy metabolism and causing accumulation of reactive oxygen species (ROS). Furthermore, transcriptome analysis of B. cinerea following exposure to VOCs revealed 4590 differentially expressed genes (DEGs) (1388 upregulated, 3202 downregulated). Through GO analysis, these DEGs were determined to be enriched in intrinsic components of membrane, integral components of membrane, and membrane parts, while KEGG analysis indicated that they were enriched in many amino acid metabolism pathways. Significantly, the DEGs related to ergosterol biosynthesis, ATPase, mitochondrial respiratory chain, malate dehydrogenase, and cell membrane showed down-regulation, corroborating the biochemical analyses. Taken together, these results suggest that the antifungal activity of P. fluorescens ZX-producing VOCs against B. cinerea occurs primary mechanisms: causing significant damage to the cell membrane, negatively affecting respiration, and interfering with amino acid metabolism.

Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates / Produção significativa de vanilina e amplificação in vitro do gene ech em isolados bacterianos locais

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.

eDNA, Amyloid Fibers and Membrane Vesicles Identified in Pseudomonas fluorescens SBW25 Biofilms.

Pseudomonas fluorescens SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air-liquid interface biofilms in experimental microcosms and on plant surfaces. Previous investigations have shown that cellulose is the primary structural matrix component in the robust and well-attached Wrinkly Spreader biofilm, as well as in the fragile Viscous Mass biofilm. Here, we demonstrate that both biofilms include extracellular DNA (eDNA) which can be visualized using confocal laser scanning microscopy (CLSM), quantified by absorbance measurements, and degraded by DNase I treatment. This eDNA plays an important role in cell attachment and biofilm development. However, exogenous high-molecular-weight DNA appears to decrease the strength and attachment levels of mature Wrinkly Spreader biofilms, whereas low-molecular-weight DNA appears to have little effect. Further investigation with CLSM using an amyloid-specific fluorophore suggests that the Wrinkly Spreader biofilm might also include Fap fibers, which might be involved in attachment and contribute to biofilm strength. The robust nature of the Wrinkly Spreader biofilm also allowed us, using MALDI-TOF mass spectrometry, to identify matrix-associated proteins unable to diffuse out of the structure, as well as membrane vesicles which had a different protein profile compared to the matrix-associated proteins. CLSM and DNase I treatment suggest that some vesicles were also associated with eDNA. These findings add to our understanding of the matrix components in this model pseudomonad, and, as found in other biofilms, biofilm-specific products and material from lysed cells contribute to these structures through a range of complex interactions.

Metabolic adaptation and ATP homeostasis in Pseudomonas fluorescens exposed to phosphate stress.

Phosphate (Pi) is essential for life as it is an integral part of the universal chemical energy adenosine triphosphate (ATP), and macromolecules such as, DNA, RNA proteins and lipids. Despite the core roles and the need of this nutrient in living cells, some bacteria can grow in environments that are poor in Pi. The metabolic mechanisms that enable bacteria to proliferate in a low phosphate environment are not fully understood. In this study, the soil microbe Pseudomonas (P.) fluorescens was cultured in a control and a low Pi (stress) medium in order to delineate how energy homeostasis is maintained. Although there was no significant variation in biomass yield in these cultures, metabolites like isocitrate, oxaloacetate, pyruvate and phosphoenolpyruvate (PEP) were markedly increased in the phosphate-starved condition. Components of the glycolytic, glyoxylate and tricarboxylic acid cycles operated in tandem to generate ATP by substrate level phosphorylation (SLP) as NADH-producing enzymes were impeded. The α-ketoglutarate (KG) produced when glutamine, the sole carbon nutrient was transformed into phosphoenol pyruvate (PEP) and succinyl-CoA (SC), two high energy moieties. The metabolic reprogramming orchestrated by isocitrate lyase (ICL), phosphoenolpyruvate synthase (PEPS), pyruvate phosphate dikinase (PPDK), and succinyl-CoA synthetase fulfilled the ATP budget. Cell free extract experiments confirmed ATP synthesis in the presence of such substrates as PEP, oxaloacetate and isocitrate respectively. Gene expression profiling revealed elevated transcripts associated with numerous enzymes including ICL, PEPS, and succinyl-CoA synthetase (SCS). This microbial adaptation will be critical in promoting biological activity in Pi-poor ecosystems.

Spatial Structure Formation by RsmE-Regulated Extracellular Secretions in Pseudomonas fluorescens Pf0-1.

Cells in microbial communities on surfaces live and divide in close proximity, which greatly enhances the potential for social interactions. Spatiogenetic structures are manifested through competitive and cooperative interactions among the same and different genotypes within a shared space, and extracellular secretions appear to function dynamically at the forefront. A previous experimental evolution study utilizing Pseudomonas fluorescens Pf0-1 colonies demonstrated that diverse mutations in the rsmE gene were repeatedly and exclusively selected through the formation of a dominant spatial structure. RsmE's primary molecular function is translation repression, and its homologs regulate various social and virulence phenotypes. Pseudomonas spp. possess multiple paralogs of Rsm proteins, and RsmA, RsmE, and RsmI are the most prevalent. Here, we demonstrate that the production of a mucoid polymer and a biosurfactant are exclusively regulated through RsmE, contradicting the generalized notion of functional redundancy among the Rsm paralogs. Furthermore, we identified the biosurfactant as the cyclic lipopeptide gacamide A. Competition and microscopy analyses showed that the mucoid polymer is solely responsible for creating a space of low cellular density, which is shared exclusively by the same genotype. Gacamide A and other RsmE-regulated products appear to establish a physical boundary that prevents the encroachment of the competing genotype into the newly created space. Although cyclic lipopeptides and other biosurfactants are best known for their antimicrobial properties and reducing surface tension to promote the spreading of cells on various surfaces, they also appear to help define spatial structure formation within a dense community. IMPORTANCE In densely populated colonies of the bacterium Pseudomonas fluorescens Pf0-1, diverse mutations in the rsmE gene are naturally selected by solving the problem of overcrowding. Here, we show that RsmE-regulated secretions function together to create and protect space of low cell density. A biosurfactant generally promotes the spreading of bacterial cells on abiotic surfaces; however, it appears to function atypically within a crowded population by physically defining genotypic boundaries. Another significant finding is that these secretions are not regulated by RsmE's paralogs that share high sequence similarity. The experimental pipeline described in this study is highly tractable and should facilitate future studies to explore additional RsmE-regulated products and address why RsmE is functionally unique from its paralogs.

The Regulatory Network Involving PcoR, RsaL, and MvaT Coordinates the Quorum-Sensing System in Pseudomonas fluorescens 2P24.

Pseudomonas fluorescens 2P24 is a beneficial plant root-associated microorganism capable of suppressing several soilborne plant diseases. The capacity of P. fluorescens to aggressively colonize the rhizosphere is an important requirement for its biocontrol trait. We previously found that the PcoI/PcoR quorum-sensing system (QS) is involved in regulating the rhizosphere colonization of P. fluorescens. Here, we revealed a sophisticated regulatory network that connects PcoR, RsaL, and MvaT proteins to fine-tune the PcoI/PcoR QS system. Our data showed that PcoR could directly bind to the promoter region of pcoI thereby inducing the PcoI/PcoR QS system, whereas RsaL binds simultaneously with PcoR to the promoter region of pcoI and represses the PcoR-dependent activation of pcoI gene. In addition, RsaL indirectly downregulates the expression of pcoR. Furthermore, we showed that disruption of mvaT enhanced the expression of pcoI, pcoR, and rsaL, whereas MvaT controls the PcoI/PcoR QS in a RsaL-independent manner. Overall, this study elucidates that PcoR, RsaL, and MvaT regulate the PcoI/PcoR QS through a multi-tiered regulatory mechanism and that PcoR is necessary in the RsaL- and MvaT-mediated repression on the expression of pcoI. IMPORTANCE The PcoI/PcoR quorum-sensing system of Pseudomonas fluorescens 2P24 is important for its effective colonization in the plant rhizosphere. Many regulatory elements appear to directly or indirectly influence the QS system. Here, we found a complex regulatory network employing transcriptional factors PcoR, RsaL, and MvaT to influence the expression of the PcoI/PcoR QS in P. fluorescens 2P24. Our results indicate that PcoR and RsaL directly bind to the promoter region of pcoI and then positively and negatively regulate the expression of pcoI, respectively. Furthermore, the H-NS family protein MvaT negatively controls the PcoI/PcoR QS in a RsaL-independent manner. Taken together, our data provide new insights into the interplays between different regulatory elements that fine-tune the QS system of P. fluorescens.

Molecular basis for coordinating secondary metabolite production by bacterial and plant signaling molecules.

The production of secondary metabolites is a major mechanism used by beneficial rhizobacteria to antagonize plant pathogens. These bacteria have evolved to coordinate the production of different secondary metabolites due to the heavy metabolic burden imposed by secondary metabolism. However, for most secondary metabolites produced by bacteria, it is not known how their biosynthesis is coordinated. Here, we showed that PhlH from the rhizobacterium Pseudomonas fluorescens is a TetR-family regulator coordinating the expression of enzymes related to the biosynthesis of several secondary metabolites, including 2,4-diacetylphloroglucinol (2,4-DAPG), mupirocin, and pyoverdine. We present structures of PhlH in both its apo form and 2,4-DAPG-bound form and elucidate its ligand-recognizing and allosteric switching mechanisms. Moreover, we found that dissociation of 2,4-DAPG from the ligand-binding domain of PhlH was sufficient to allosterically trigger a pendulum-like movement of the DNA-binding domains within the PhlH dimer, leading to a closed-to-open conformational transition. Finally, molecular dynamics simulations confirmed that two distinct conformational states were stabilized by specific hydrogen bonding interactions and that disruption of these hydrogen bonds had profound effects on the conformational transition. Our findings not only reveal a well-conserved route of allosteric signal transduction in TetR-family regulators but also provide novel mechanistic insights into bacterial metabolic coregulation.

Gaseous NO2 induces various envelope alterations in Pseudomonas fluorescens MFAF76a.

Anthropogenic atmospheric pollution and immune response regularly expose bacteria to toxic nitrogen oxides such as NO• and NO2. These reactive molecules can damage a wide variety of biomolecules such as DNA, proteins and lipids. Several components of the bacterial envelope are susceptible to be damaged by reactive nitrogen species. Furthermore, the hydrophobic core of the membranes favors the reactivity of nitrogen oxides with other molecules, making membranes an important factor in the chemistry of nitrosative stress. Since bacteria are often exposed to endogenous or exogenous nitrogen oxides, they have acquired protection mechanisms against the deleterious effects of these molecules. By exposing bacteria to gaseous NO2, this work aims to analyze the physiological effects of NO2 on the cell envelope of the airborne bacterium Pseudomonas fluorescens MFAF76a and its potential adaptive responses. Electron microscopy showed that exposure to NO2 leads to morphological alterations of the cell envelope. Furthermore, the proteomic profiling data revealed that these cell envelope alterations might be partly explained by modifications of the synthesis pathways of multiple cell envelope components, such as peptidoglycan, lipid A, and phospholipids. Together these results provide important insights into the potential adaptive responses to NO2 exposure in P. fluorescens MFAF76a needing further investigations.

Rice straw enhancing catalysis of Pseudomonas fluorescens lipase for synthesis of citronellyl acetate.

Citronellyl acetate as an important flavor, can be effectively synthesized by lipase catalysis in nonaqueous system. But lipases usually behave low catalytic activity due to aggregation and denaturation of them in organic phase. To enhance the nonaqueous catalysis, based on the mechanism of lipases activated at water/oil (organic phase) interface, the inexpensive race straw was processed into powder and filaments on which Pseudomonas fluorescens lipase was immobilized by physical adsorption, used for synthesis of citronellyl acetate via transesterification of citronellol and vinyl acetate. Results showed that the desired loading was 10 mg lipase immobilized on 30 mg rice straw filaments or 25 mg rice straw powder. When the two immobilized lipases were employed in the reaction system consisted of 1-mL citronellol and 2-mL vinyl acetate at 37 â„ƒ and 160 rpm, the conversions all reached 99.8% after 12 h. Under the reaction condition, the conversion catalyzed by 10 mg native lipase was 85.1%. Undergoing six times of 8-h reuses in the organic system, the filament and power immobilized lipases had weak activity attenuation rates 0.36 and 0.32% h-1, lower than 1.52% h-1 of native lipase. Even at the room temperature and the static state without shaking and stirring, the rice straw filaments immobilized lipase could brought conversion 62.9% after 10 h but the native lipase only gave 37.0%. Obviously, the rice straw, especially its filaments, is an inexpensive and available natural material to prepare immobilized lipase with desired catalysis in organic phase, meant significant potential in flavor industry.