Biofilm as a production platform for heterologous production of rhamnolipids by the non‑pathogenic strain <i>Pseudomonas</i> <i>putida</i> KT2440

BACKGROUND: Although a transition toward sustainable production of chemicals is needed, the physiochemical properties of certain biochemicals such as biosurfactants make them challenging to produce in conventional bioreactor systems. Alternative production platforms such as surface-attached biofilm populations could potentially overcome these challenges. Rhamnolipids are a group of biosurfactants highly relevant for industrial applications. However, they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa using hydrophobic substrates such as plant oils. As the biosynthesis is tightly regulated in P. aeruginosa a heterologous production of rhamnolipids in a safe organism can relive the production from many of these limitations and alternative substrates could be used. RESULTS: In the present study, heterologous production of biosurfactants was investigated using rhamnolipids as the model compound in biofilm encased Pseudomonas putida KT2440. The rhlAB operon from P. aeruginosa was introduced into P. putida to produce mono-rhamnolipids. A synthetic promoter library was used in order to bypass the normal regulation of rhamnolipid synthesis and to provide varying expression levels of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology. Quantitative and structural investigations of the produced rhamnolipids were made by ultra performance liquid chromatography combined with high resolution mass spectrometry (HRMS) and tandem HRMS. The predominant rhamnolipid congener produced by the heterologous P. putida biofilm was mono-rhamnolipid with two C(10) fatty acids. CONCLUSION: This study shows a successful application of synthetic promoter library in P. putida KT2440 and a heterologous biosynthesis of rhamnolipids in biofilm encased cells without hampering biofilm capabilities. These findings expands the possibilities of cultivation setups and paves the way for employing biofilm flow systems as production platforms for biochemicals, which as a consequence of physiochemical properties are troublesome to produce in conventional fermenter setups, or for production of compounds which are inhibitory or toxic to the production organisms. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-016-0581-9) contains supplementary material, which is available to authorized users

Evaluation of Enoyl-Acyl Carrier Protein Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents

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Few multiyear precipitation-reduction experiments find a shift in the productivity-precipitation relationship

Well-defined productivity–precipitation relationships of ecosystems are needed as benchmarks for the validation of land models used for future projections. The productivity–precipitation relationship may be studied in two ways: the spatial approach relates differences in productivity to those in precipitation among sites along a precipitation gradient (the spatial fit, with a steeper slope); the temporal approach relates interannual productivity changes to variation in precipitation within sites (the temporal fits, with flatter slopes). Precipitation–reduction experiments in natural ecosystems represent a complement to the fits, because they can reduce precipitation below the natural range and are thus well suited to study potential effects of climate drying. Here, we analyse the effects of dry treatments in eleven multiyear precipitation–manipulation experiments, focusing on changes in the temporal fit. We expected that structural changes in the dry treatments would occur in some experiments, thereby reducing the intercept of the temporal fit and displacing the productivity–precipitation relationship downward the spatial fit. The majority of experiments (72%) showed that dry treatments did not alter the temporal fit. This implies that current temporal fits are to be preferred over the spatial fit to benchmark land-model projections of productivity under future climate within the precipitation ranges covered by the experiments. Moreover, in two experiments, the intercept of the temporal fit unexpectedly increased due to mechanisms that reduced either water loss or nutrient loss. The expected decrease of the intercept was observed in only one experiment, and only when distinguishing between the late and the early phases of the experiment. This implies that we currently do not know at which precipitation–reduction level or at which experimental duration structural changes will start to alter ecosystem productivity. Our study highlights the need for experiments with multiple, including more extreme, dry treatments, to identify the precipitation boundaries within which the current temporal fits remain valid

A Library of Theoretical Ultraviolet Spectra of Massive, Hot Stars for Evolutionary Synthesis

We computed a comprehensive set of theoretical ultraviolet spectra of hot, massive stars with the radiation-hydrodynamics code WM-Basic. This model atmosphere and spectral synthesis code is optimized for computing the strong P Cygni-type lines originating in the winds of hot stars, which are the strongest features in the ultraviolet spectral region. The computed set is suitable as a spectral library for inclusion in evolutionary synthesis models of star clusters and star-forming galaxies. The chosen stellar parameters cover the upper left Hertzsprung-Russell diagram at L >~ 10^2.75 Lsun and T_eff >~ 20,000 K. The adopted elemental abundances are 0.05 Zsun, 0.2 Zsun, 0.4 Zsun, Zsun, and 2 Zsun. The spectra cover the wavelength range from 900 to 3000 {\AA} and have a resolution of 0.4 {\AA}. We compared the theoretical spectra to data of individual hot stars in the Galaxy and the Magellanic Clouds obtained with the International Ultraviolet Explorer (IUE) and Far Ultraviolet Spectroscopic Explorer (FUSE) satellites and found very good agreement. We built a library with the set of spectra and implemented it into the evolutionary synthesis code Starburst99 where it complements and extends the existing empirical library towards lower chemical abundances. Comparison of population synthesis models at solar and near-solar composition demonstrates consistency between synthetic spectra generated with either library. We discuss the potential of the new library for the interpretation of the rest-frame ultraviolet spectra of star-forming galaxies. Properties that can be addressed with the models include ages, initial mass function, and heavy-element abundance. The library can be obtained both individually or as part of the Starburst99 package.Comment: ApJS (in press); 90 pages, 33 figures, 7 table

Differential bacterial capture and transport preferences facilitate co-growth on dietary xylan in the human gut

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Critical review on biofilm methods

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.The authors would like to acknowledge the support from the EU COST Action BacFoodNet FA1202

Biofilm Induced Tolerance towards Antimicrobial Peptides

Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms