The dynamic architecture of the metabolic switch in Streptomyces coelicolor

[EN] Background: During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.Results: Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.Conclusions: Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological settingSIWe are very grateful to Mervyn Bibb for his generous support with the Affymetrix custom microarray design. We acknowledge the excellent technical help of K. Klein, S. Poths, M. Walter, A. Øverby and E. Hansen. This project was supported by grants of the ERA-NET SySMO Project [GEN2006-27745-E/SYS]: (P-UK-01-11-3i) and the Research Council of Norway [project no. 181840/I30

Factors associated with internalizing or somatic symptoms in a cross-sectional study of school children in grades 1-10

<p>Abstract</p> <p>Background</p> <p>School related factors that may contribute to children's subjective health have not been extensively studied. We assessed whether factors assumed to promote health and factors assumed to have adverse effects were associated with self-reported internalizing or somatic symptoms.</p> <p>Methods</p> <p>In a cross-sectional study, 230 boys and 189 girls in grades 1-10 from five schools responded to the same set of questions. Proportional odds logistic regression was used to assess associations of school related factors with the prevalence of sadness, anxiety, stomach ache, and headache.</p> <p>Results</p> <p>In multivariable analyses, perceived loneliness showed strong and positive associations with sadness (odds ratio, 1.94, 95% CI 1.42 to 2.64), anxiety (odds ratio, 1.78, 95% CI 1.31 to 2.42), and headache (odds ratio, 1.47, 95% CI 1.10 to 1.96), with consistently stronger associations for girls than boys. Among assumed health promoting factors, receiving necessary help from teachers was associated with lower prevalence of stomach ache in girls (odds ratio, 0.51, 95% CI 0.30 to 0.87).</p> <p>Conclusions</p> <p>These findings suggest that perceived loneliness may be strongly related to both internalizing and somatic symptoms among school children, and for girls, the associations of loneliness appear to be particularly strong.</p

Pros and cons of different therapeutic antibody formats for recombinant antivenom development.

Antibody technologies are being increasingly applied in the field of toxinology. Fuelled by the many advances in immunology, synthetic biology, and antibody research, different approaches and antibody formats are being investigated for the ability to neutralize animal toxins. These different molecular formats each have their own therapeutic characteristics. In this review, we provide an overview of the advances made in the development of toxin-targeting antibodies, and discuss the benefits and drawbacks of different antibody formats in relation to their ability to neutralize toxins, pharmacokinetic features, propensity to cause adverse reactions, formulation, and expression for research and development (R&D) purposes and large-scale manufacturing. A research trend seems to be emerging towards the use of human antibody formats as well as camelid heavy-domain antibody fragments due to their compatibility with the human immune system, beneficial therapeutic properties, and the ability to manufacture these molecules cost-effectively

Rapid reagentless quantification of alginate biosynthesis in Pseudomonas fluorescens bacteria mutants using FT-IR spectroscopy coupled to multivariate partial least squares regression

Alginate is an important medical and commercial product and currently is isolated from seaweeds. Certain microorganisms also produce alginate and these polymers have the potential to replace seaweed alginates in some applications, mainly because such production will allow much better and more reproducible control of critical qualitative polymer properties. The research conducted here presents the development of a new approach to this problem by analysing a transposon insertion mutant library constructed in an alginate-producing derivative of the Pseudomonas fluorescens strain SBW25. The procedure is based on the non-destructive and reagent-free method of Fourier transform infrared (FT-IR) spectroscopy which is used to generate a complex biochemical infrared fingerprint of the medium after bacterial growth. First, we investigate the potential differences caused by the growth media fructose and glycerol on the bacterial phenotype and alginate synthesis in 193 selected P. fluorescens mutants and show that clear phenotypic differences are observed in the infrared fingerprints. In order to quantify the level of the alginate we also report the construction and interpretation of multivariate partial least squares regression models which were able to quantify alginate levels successfully with typical normalized root-mean-square error in predictions of only approximately 14 %. We have demonstrated that this high-throughput approach can be implemented in alginate screens and we believe that this FT-IR spectroscopic methodology, when combined with the most appropriate chemometrics, could easily be modified for the quantification of other valuable microbial products and play a valuable screening role for synthetic biology

The dynamic architecture of the metabolic switch in Streptomyces coelicolor

Background: During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples. Results: Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis. Conclusions: Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting

Investigating alginate production and carbon utilization in Pseudomonas fluorescens SBW25 using mass spectrometry-based metabolic profiling

Metabolic profiling of Pseudomonas fluorescens SBW25 and various mutants derived thereof was performed to explore how the bacterium adapt to changes in carbon source and upon induction of alginate synthesis. The experiments were performed at steady-state conditions in nitrogen-limited chemostats using either fructose or glycerol as carbon source. Carbon source consumption was up-regulated in the alginate producing mutant with inactivated anti-sigma factor MucA. The mucA- mutants (also non-alginate producing mucA- control strains) had a higher dry weight yield on carbon source implying a change in carbon and energy metabolism due to the inactivation of the anti-sigma factor MucA. Both LC–MS/MS and GC–MS methods were used for quantitative metabolic profiling, and major reorganization of primary metabolite pools in both an alginate producing and a carbon source dependent manner was observed. Generally, larger changes were observed among the phosphorylated glycolytic metabolites, the pentose phosphate pathway metabolites and the nucleotide pool than among amino acids and citric acid cycle compounds. The most significant observation at the metabolite level was the significantly reduced energy charge of the mucA- mutants (both alginate producing and non-producing control strains) compared to the wild type strain. This reduction was caused more by a strong increase in the AMP pool than changes in the ATP and ADP pools. The alginate-producing mucA- mutant had a slightly increased GTP pool, while the GDP and GMP pools were strongly increased compared to non-producing mucA- strains and to the wild type. Thus, whilst changes in the adenosine phosphate nucleotide pool are attributed to the mucA inactivation, adjustments in the guanosine phosphate nucleotide pool are consequences of the GTP-dependent alginate production induced by the mucA inactivation. This metabolic profiling study provides new insight into carbon and energy metabolism of the alginate producer P. fluorescens

Mapping global effects of the anti-sigma factor MucA in Pseudomonas fluorescens SBW25 through genome-scale metabolic modeling

BackgroundAlginate is an industrially important polysaccharide, currently produced commercially by harvesting of marine brown sea-weeds. The polymer is also synthesized as an exo-polysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter, and these organisms may represent an alternative alginate source in the future. The current work describes an attempt to rationally develop a biological system tuned for very high levels of alginate production, based on a fundamental understanding of the system through metabolic modeling supported by transcriptomics studies and carefully controlled fermentations.ResultsAlginate biosynthesis in Pseudomonas fluorescens was studied in a genomics perspective, using an alginate over-producing strain carrying a mutation in the anti-sigma factor gene mucA. Cells were cultivated in chemostats under nitrogen limitation on fructose or glycerol as carbon sources, and cell mass, growth rate, sugar uptake, alginate and CO2 production were monitored. In addition a genome scale metabolic model was constructed and samples were collected for transcriptome analyses. The analyses show that polymer production operates in a close to optimal way with respect to stoichiometric utilization of the carbon source and that the cells increase the uptake of carbon source to compensate for the additional needs following from alginate synthesis. The transcriptome studies show that in the presence of the mucA mutation, the alg operon is upregulated together with genes involved in energy generation, genes on both sides of the succinate node of the TCA cycle and genes encoding ribosomal and other translation-related proteins. Strains expressing a functional MucA protein (no alginate production) synthesize cellular biomass in an inefficient way, apparently due to a cycle that involves oxidation of NADPH without ATP production. The results of this study indicate that the most efficient way of using a mucA mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation.ConclusionsThe insights gained in this study should be very useful for a future efficient production of microbial alginates

Molecular insights into alginate β‐lactoglobulin A multivalencies – the foundation for their amorphous aggregates and coacervation

For improved control of biomaterial property design, a better understanding of complex coacervation involving anionic polysaccharides and proteins is needed. Here, we address the initial steps in condensate formation of β‐lactoglobulin A (β‐LgA) with nine defined alginate oligosaccharides (AOSs) and describe their multivalent interactions in structural detail. Binding of AOSs containing four, five, or six uronic acid residues (UARs), either all mannuronate (M), all guluronate (G), or alternating M and G embodying the block structural components of alginates, was characterized by isothermal titration calorimetry, nuclear magnetic resonance spectroscopy (NMR), and molecular docking. β‐LgA was highly multivalent exhibiting binding stoichiometries decreasing from five to two AOSs with increasing degree of polymerization (DP) and similar affinities in the mid micromolar range. The different AOS binding sites on β‐LgA were identified by NMR chemical shift perturbation analyses and showed diverse compositions of charged, polar and hydrophobic residues. Distinct sites for the shorter AOSs merged to accommodate longer AOSs. The AOSs bound dynamically to β‐LgA, as concluded from saturation transfer difference and (1)H‐ligand‐targeted NMR analyses. Molecular docking using Glide within the Schrödinger suite 2016‐1 revealed the orientation of AOSs to only vary slightly at the preferred β‐LgA binding site resulting in similar XP glide scores. The multivalency coupled with highly dynamic AOS binding with lack of confined conformations in the β‐LgA complexes may help explain the first steps toward disordered β‐LgA alginate coacervate structures

The Effect of Prolonged Use of a Wearable Soft-Robotic Glove Post Stroke -: A Proof-of-Principle

Many stroke survivors encounter difficulties in the performance of activities of daily life due to limitations in functional use of the hand. Robotic technology has the potential to compensate for this loss by providing the support that is required to perform activities of daily living, especially when these devices are wearable comfortably for many hours at home. As a first step towards the implementation of assistive technology in the homes of stroke survivors, usability along with the potential effect of prolonged use of a wearable soft-robotic glove during activities of daily life on functional task performance was assessed in this study. Therefore, five chronic stroke survivors were asked to use a wearable soft-robotic glove for four weeks at home during preferred activities of daily life. Before and after the home use of the glove, functional task performance was assessed in a lab environment. After the use of the glove, system usability was assessed. The prolonged use of the glove resulted in an improved supported and unsupported functional performance during tasks related to activities of daily life, as measured with the Jebsen-Taylor Hand Function Test. Promising system usability results were found indicating a good probability for acceptance of the glove. The results from this study indicate the potential of the current glove to be used as assistive tool, which even showed a therapeutic effect. Yet, the glove should be tested in a larger sample for better interpretation and confirmation of these promising results