Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface-binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection. IMPORTANCE With the currently available antibiotics, the treatment of TB takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms

Elevated atmospheric [CO₂] can dramatically increase wheat yields in semi-arid environments and buffer against heat waves

Tausz, M ORCiD: 0000-0001-8205-8561Wheat production will be impacted by increasing concentration of atmospheric CO2 [CO2], which is expected to rise from about 400 μmol mol-1 in 2015 to 550 μmol mol-1 by 2050. Changes to plant physiology and crop responses from elevated [CO2] (e[CO2]) are well documented for some environments, but field-level responses in dryland Mediterranean environments with terminal drought and heat waves are scarce. The Australian Grains Free Air CO2 Enrichment facility was established to compare wheat (Triticum aestivum) growth and yield under ambient (~370 μmol-1 in 2007) and e[CO2] (550 μmol-1) in semi-arid environments. Experiments were undertaken at two dryland sites (Horsham and Walpeup) across three years with two cultivars, two sowing times and two irrigation treatments. Mean yield stimulation due to e[CO2] was 24% at Horsham and 53% at Walpeup, with some treatment responses greater than 70%, depending on environment. Under supplemental irrigation, e[CO2] stimulated yields at Horsham by 37% compared to 13% under rainfed conditions, showing that water limited growth and yield response to e[CO2]. Heat wave effects were ameliorated under e[CO2] as shown by reductions of 31% and 54% in screenings and 10% and 12% larger kernels (Horsham and Walpeup). Greatest yield stimulations occurred in the e[CO2] late sowing and heat stressed treatments, when supplied with more water. There were no clear differences in cultivar response due to e[CO2]. Multiple regression showed that yield response to e[CO2] depended on temperatures and water availability before and after anthesis. Thus, timing of temperature and water and the crop's ability to translocate carbohydrates to the grain postanthesis were all important in determining the e[CO2] response. The large responses to e[CO2] under dryland conditions have not been previously reported and underscore the need for field level research to provide mechanistic understanding for adapting crops to a changing climate. © 2016 John Wiley & Sons Ltd

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface–binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection.Peer reviewe

Recognition of T-rich single-stranded DNA by the cold shock protein Bs-CspB in solution

Cold shock proteins (CSP) belong to the family of single-stranded nucleic acid binding proteins with OB-fold. CSP are believed to function as ‘RNA chaperones’ and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT(7)) by NMR spectroscopy. Bs-CspB reveals an almost invariant conformation when bound to dT(7) with only minor reorientations in loop β1–β2 and β3–β4 and of few aromatic side chains involved in base stacking. Binding studies of protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. (15)N NMR relaxation and H/D exchange experiments revealed that binding of dT(7) increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire protein and especially of loop β3–β4

Phenology Modelling and Forest Disturbance Mapping with Sentinel-2 Time Series in Austria

Worldwide, forests provide natural resources and ecosystem services. However, forest ecosystems are threatened by increasing forest disturbance dynamics, caused by direct human activities or by altering environmental conditions. It is decisive to reconstruct and trace the intra- to transannual dynamics of forest ecosystems. National to local forest authorities and other stakeholders request detailed area-wide maps that delineate forest disturbance dynamics at various spatial scales. We developed a time series analysis (TSA) framework that comprises data download, data management, image preprocessing and an advanced but flexible TSA. We use dense Sentinel-2 time series and a dynamic Savitzky–Golay-filtering approach to model robust but sensitive phenology courses. Deviations from the phenology models are used to derive detailed spatiotemporal information on forest disturbances. In a first case study, we apply the TSA to map forest disturbances directly or indirectly linked to recurring bark beetle infestation in Northern Austria. In addition to spatially detailed maps, zonal statistics on different spatial scales provide aggregated information on the extent of forest disturbances between 2018 and 2019. The outcomes are (a) area-wide consistent data of individual phenology models and deduced phenology metrics for Austrian forests and (b) operational forest disturbance maps, useful to investigate and monitor forest disturbances to facilitate sustainable forest management

FitAQ

Yet another R package to analyse the relationship between photosynthetic assimilation rate and light (light response curve). Fits a non-rectangular hyperbola of the form A ~ ((phi * Q + Amax - sqrt((phi * Q + Amax)^2 - 4 * theta * phi * Q * Amax)). A = assimilation rate, Q = PPFD, light level, phi = initial slope, theta = curvature, and Rd = respiration

Markus Löw's Quick Files

The Quick Files feature was discontinued and it’s files were migrated into this Project on March 11, 2022. The file URL’s will still resolve properly, and the Quick Files logs are available in the Project’s Recent Activity