Siderophore cooperation of the bacterium Pseudomonas fluorescens in soil

While social interactions play an important role for the evolution of bacterial siderophore production in vitro, the extent to which siderophore production is a social trait in natural populations is less clear. Here, we demonstrate that siderophores act as public goods in a natural physical environment of Pseudomonas fluorescens: soil-based compost. We show that monocultures of siderophore producers grow better than non-producers in soil, but non-producers can exploit others'' siderophores, as shown by non-producers'' ability to invade populations of producers when rare. Despite this rare advantage, non-producers were unable to outcompete producers, suggesting that producers and non-producers may stably coexist in soil. Such coexistence is predicted to arise from the spatial structure associated with soil, and this is supported by increased fitness of non-producers when grown in a shaken soil–water mix. Our results suggest that both producers and non-producers should be observed in soil, as has been observed in marine environments and in clinical populations.     

Cell-free translation system from Drosophila S2 cells that recapitulates RNAi

RNA interference (RNAi) is a fundamental mechanism of gene regulation in a variety of organisms. In Drosophila cells, long double-stranded RNAs (dsRNAs) are processed into 21- to 23-nucleotide double-stranded fragments, termed short interfering RNAs (siRNAs). The siRNAs trigger sequence-specific mRNA degradation, which results in the inhibition of gene expression. These phenomena can be recapitulated in vitro in lysates of Drosophila syncytial blastoderm embryos. In the present work, we used the common Drosophila cell line, Schneider Line 2 (S2), as a source to establish a cell-free translation system. We demonstrate here that the S2 cell-free translation system can recapitulate RNAi. Both long dsRNAs and siRNAs can trigger RNAi in this system, and the silencing effects are significant. This system should provide an important tool for biochemical analyses of the RNAi mechanism.     

Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems

Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18-159 kDa were tested for expression in a bacterial cell-free system. Fifty-one of the 63 proteins could be expressed and partially purified under denaturing conditions. Most of the expressed proteins showed yields greater than 500 ng after a single affinity purification step from 50 microl in vitro protein synthesis reactions. The in vitro protein expression plus purification in a 96-well format and analysis of the proteins by SDS-PAGE were performed by one person in 4 h. A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step affinity purification offers a rapid, efficient alternative for the high-throughput screening of clones for protein expression and solubility.     

Structure,chemistry, and biological activity of pseudophomins A and B,new cyclic lipodepsipeptides isolated from the biocontrol bacterium Pseudomonas fluorescens

Pseudophomins A and B are cyclic lipodepsipeptides isolated from Pseudomonas fluorescens strain BRG100, a bacterium with potential application for biocontrol of plant pathogens and weeds. Their chemical structures were established by a combination of spectroscopic data, X-ray crystallography, and selective chemical degradation. This unique chemical degradation allowed the unambiguous determination of the absolute configuration of the amino acid residue Leu-1, due to gamma-lactam formation followed by selective cleavage of the adjacent N(8)-C(7) bond. To the best of our knowledge this is the first application of gamma-lactam formation to the determination of absolute configuration of an adjacent amino acid. Pseudophomin B showed higher antifungal activity against the phytopathogens Phoma lingam/Leptosphaeria maculans and Sclerotinia sclerotiorum than pseudophomin A, and is likely to be the main component responsible for the antifungal activity of EtOAc extracts of strain BRG100. By contrast, pseudophomin A showed stronger inhibition of green foxtail (Setaria viridis) root germination than pseudophomin B.     

Engine out of the chassis: Cell-free protein synthesis and its uses

The translation machinery is the engine of life. Extracting the cytoplasmic milieu from a cell affords a lysate capable of producing proteins in concentrations reaching to tens of micromolar. Such lysates, derivable from a variety of cells, allow the facile addition and subtraction of components that are directly or indirectly related to the translation machinery and/or the over-expressed protein. The flexible nature of such cell-free expression systems, when coupled with high throughput monitoring, can be especially suitable for protein engineering studies, allowing one to bypass multiple steps typically required using conventional in vivo protein expression.     

Horizontal and vertical movement of Pseudomonas fluorescens toward exudate of Macrophomina phaseolina in soil: influence of motility and soil properties

The role of motility and cell surface hydrophobicity in transport and dispersal of Pseudomonas fluorescens strains LAM1-hydrophilic, LAM2-hydrophobic and LAM(NM) (non-motile mutant of LAM2) under different soil conditions was studied. Maximum adhesion was recorded for LAM2 in clay loam (70%), followed by sandy loam (68%) and sandy soil (40%). Vertical migration of P fluorescens isolates in soils was recorded at 5 and 25 cm flow of wafer or M. phaseolina exudate. In all the treatments, LAM1 exhibited maximum migration followed, by LAM2 and LAM(NM). The rate of migration of such isolates was lowered in water irrigated soils compared to those irrigated with M. phaseolina exudate. In sandy soil, cells of LAM1 migrated up to 13 cm in comparison to LAM2 (11 cm) and LAN(NM) (9 cm) at 5 cm flow of fungal exudate. Population of LAM1, LAM2 and LAM(NM) was 5.7, 5.68 and 5.61 log cfu g(-1) soil at 1 cm depth, but it decreased to 2.56, 2.21 and 1.99 log cfu during migration up to 11 cm in sandy soil at 5 cm flow of fungal exudate. Greater motility was observed in sandy soil irrigated with water or fungal exudate, followed by sandy loam and clay loam. In general, filtration coefficient (lambda) of P. fluorescens was higher in soils irrigated with 5 cm of water or exudate than with 25 cm of irrigation. The horizontal movement of P. fluorescens strains in sandy soil adjusted at different psi m showed marked reduction with decrease in psi m. The non-motile LAN(NM) did not show chemotactic response and migrated up to a maximum of 3 mm in saturated soils (0 kPa). After 96 h, LAM1 and LAM2 migrated upto 35 and 29 mm respectively in sandy soil. Motile isolates had significantly greater colonization of M. phaseolina sclerotia over the non-motile mutant.     

Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions

The functional stability of mRNA is one of the crucial factors affecting the efficiency of cell-free protein synthesis. The importance of the stability of mRNA in the prolonged synthesis of protein molecules becomes even greater when the cell-free protein synthesis is directed by PCR-amplified DNAs, because the linear DNAs are rapidly degraded by the endogenous nucleases and, thus, the continuous generation of mRNA molecules is limited. With the aim of developing a highly efficient cell-free protein synthesis system directed by PCR products, in this study, we describe a systematic approach to enhance the stability of mRNA in cell-free extracts. First, exonuclease-mediated degradation was substantially reduced by introducing a stem-loop structure at the 3'-end of the mRNA. The endonucleolytic cleavage of the mRNA was minimized by using an S30 extract prepared from an Escherichia coli strain that is deficient in a major endonuclease (RNase E). Taken together, through the retardation of the endonucleolytic and exonucleolytic degradations of the mRNA molecules, the level of protein expression from the PCR-amplified DNA templates becomes comparable to that of conventional plasmid-based reactions. The enhanced productivity of the PCR-based cell-free protein synthesis enables the high-throughput generation of protein molecules required for many post-genomic applications.     

Improving cell-free protein synthesis for stable-isotope labeling

Cell-free protein synthesis is suitable for stable-isotope labeling of proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-isotope labeling, although it is less productive than other systems. A system containing a high concentration of potassium l-glutamate (l-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-isotope labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium d-glutamate (d-Glu system). The productivity of the d-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the ¹H–¹⁵N HSQC spectrum of the uniformly stable-isotope labeled Ras protein, prepared with the d-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the protein produced with the d-Glu system. These results indicate that the d-Glu system is a highly productive cell-free system that is especially useful for stable-isotope labeling of proteins. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Fluorene Cometabolism by Rhodococcus rhodochrous and Pseudomonas fluorescens Cultures

The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase. Under certain cultivation conditions, fluorene was completely consumed from the medium. The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source. An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.     

Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase

Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412 μg/mL of purified GLuc, relative to 5 μg/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2×10²⁴ photons/s/mol, the highest reported activity for any characterized luciferase.     

Extracellular Protease as a Reversible Adhesion Regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2–3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h^–1, the fraction of proteins in cells being almost the same (60–70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3–5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.     

Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a biocontrol agent isolated from a wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide and siderophore(s). Our recent work revealed that strain 2P24 employs a quorum-sensing system to regulate its biocontrol activity. In this study, we identified a quorum-sensing system consisting of PcoR and PcoI of the LuxR–LuxI family from strain 2P24. Deletion of pcoI from 2P24 abolishes the production of the quorum-sensing signals, but does not detectably affect the production of antifungal metabolites. However, the mutant is significantly defective in biofilm formation, colonization on wheat rhizosphere and biocontrol ability against wheat take-all, whilst complementation of pcoI restores the biocontrol activity to the wild-type level. Our data indicate that quorum sensing is involved in regulation of biocontrol activity in P. fluorescens 2P24.     

Induction of polymyxin resistance in Pseudomonas fluorescens by phosphate limitation

Shift of Pseudomonas fluorescens NCMB 129 from a phosphate rich into a phosphate limited medium results in a reduction of the membrane phospholipids phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Concomitantly a positively charged ornithine amide lipid is synthesized. The gradual increase of this lipid is paralleled by an increasing resistance to polymyxin B. The binding capacities of intact cells, and isolated inner and outer membranes for the antibiotic are reduced in the resistant organisms. It is discussed that the observed effect could be circumstantial evidence that the positively charged polymyxin B needs negatively charged receptors in biological membranes in order to exert its antibiotic activity.List of Abbreviations PE phosphatidylethanolamine - PG phosphatidylglycerol - CL cardiolipin - PX polymyxin B     

Cell-free protein synthesis of perdeuterated proteins for NMR studies

Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins with selective isotope-labeling of selected amino acid residues on a perdeuterated background for studies of enzyme active sites or for ligand screening in drug discovery projects, as well as the synthesis of perdeuterated polypeptides for NMR spectroscopy with large supra-molecular structures. As an illustration, it is demonstrated that the 800-kDa chaperonine GroEL synthesized with the D-S30 cell-free system had a uniform deuteration level of about 95% and assembled into its biologically active oligomeric form.     

The pyoverdine from Pseudomonas chlororaphis D-TR133 showing mutual acceptance with the pyoverdine of Pseudomonas fluorescens CHA0

From Pseudomonas chlororaphis D-TR133 a pyoverdine was isolated and its primary structure were elucidated by spectroscopic methods and degradation reactions. Despite some structural differences, its Fe(III) complex and that of the pyoverdine from Pseudomonas fluorescens CHA0 were taken up by either strain with a high rate. This is explained by a structural similarity between the two pyoverdines which were shown to differ in their structures only by the replacement of Lys by Ala in the C-terminal part of the molecules. An unexpected feature is that the main pyoverdine of P. chlororaphis D-TR133 is accompanied by a minor one where specifically one Ala is replaced by Gly. So far amino acid variations in the peptide chain of pyoverdines produced by a given strain had not been observed amongst the producers of the about fifty pyoverdines reported in the literature.     

Evaluation of an Antibiotic-Producing Strain of Pseudomonas fluorescens for Suppression of Plant-Parasitic Nematodes

The antibiotic 2,4-diacetylphloroglucinol (DAPG), produced by some strains of Pseudomonas spp., is involved in suppression of several fungal root pathogens as well as plant-parasitic nematodes. The primary objective of this study was to determine whether Wood1R, a D-genotype strain of DAPG-producing P. fluorescens, suppresses numbers of both sedentary and migratory plant-parasitic nematodes. An experiment was conducted in steam-heated soil and included two seed treatments (with Wood1R and a control without the bacterium) and six plant-nematode combinations which were Meloidogyne incognita on cotton, corn, and soybean; M. arenaria on peanut; Heterodera glycines on soybean; and Paratrichodorus minor on corn. Wood 1R had no effect on final numbers of M. arenaria, P. minor, or H. glycines; however, final numbers of M. incognita were lower when seeds were treated with Wood1R than left untreated, and this reduction was consistent among host plants. Population densities of Wood1R were greater on the roots of corn than on the other crops, and the bacterium was most effective in suppressing M. incognita on corn, with an average reduction of 41%. Despite high population densities of Wood1R on corn, the bacterium was not able to suppress numbers of P. minor. When comparing the suppression of M. incognita on corn in natural and steam-heated soil, egg production by the nematode was suppressed in natural compared to steamed soil, but the presence of Wood1R did not result in additional suppression of the nematodes in the natural soil. These data indicate that P. fluorescens strain Wood1R has the capacity to inhibit some populations of plant-parasitic nematodes. However, consistent suppression of nematodes in natural soils seems unlikely.