| 1. |
- Moreno-Gamez, Stefany, et al.
(författare)
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Wide lag time distributions break a trade-off between reproduction and survival in bacteria
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:31, s. 18729-18736
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Tidskriftsartikel (refereegranskat)abstract
- Many microorganisms face a fundamental trade-off between reproduction and survival: Rapid growth boosts population size but makes microorganisms sensitive to external stressors. Here, we show that starved bacteria encountering new resources can break this trade-off by evolving phenotypic heterogeneity in lag time. We quantify the distribution of single-cell lag times of populations of starved Escherichia coli and show that population growth after starvation is primarily determined by the cells with shortest lag due to the exponential nature of bacterial population dynamics. As a consequence, cells with long lag times have no substantial effect on population growth resumption. However, we observe that these cells provide tolerance to stressors such as antibiotics. This allows an isogenic population to break the trade-off between reproduction and survival. We support this argument with an evolutionary model which shows that bacteria evolve wide lag time distributions when both rapid growth resumption and survival under stressful conditions are under selection. Our results can explain the prevalence of antibiotic tolerance by lag and demonstrate that the benefits of phenotypic heterogeneity in fluctuating environments are particularly high when minorities with extreme phenotypes dominate population dynamics.
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| 2. |
- Baumgarten, Thomas, et al.
(författare)
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Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Hjelm, Anna, et al.
(författare)
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Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) and GFP-Fusions
- 2013
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Ingår i: Membrane Biogenesis. - Totowa, USA : Humana Press. - 9781627034869 - 9781627034876 ; , s. 381-400
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Bokkapitel (refereegranskat)abstract
- Optimizing the conditions for the overexpression of membrane proteins in E. coli and their subsequent purification is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain, which conveniently allows to identify the optimal expression intensity of a membrane protein using only one strain, and membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the production of high-quality membrane protein material for functional and structural studies.
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| 4. |
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| 5. |
- Klepsch, Mirjam, 1983-, et al.
(författare)
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Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes
- 2008
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 46:2, s. 48-53
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Tidskriftsartikel (refereegranskat)abstract
- In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS–PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS–PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS–PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS–PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS–PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.
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| 6. |
- Lee, Danna, et al.
(författare)
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Internal expression of Rhs toxins allows protection against Rhs toxin delivery by stabilizing the cognate Rhs antitoxins
- 2022
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Rhs elements are some of the most positively selected genes known and were recently found to function as type II TA-systems in addition to their previous role as delivered toxins. Antitoxins of type II TA systems are often inherently unstable proteins. Here we discover that the RhsI immunity proteins in Salmonella enterica serovar Typhimurium are also unstable on their own, but stabilized in the presence of their cognate toxins. This raises interesting questions regarding how such unstable immunity proteins can protect against incoming toxins delivered by neighboring attackers. In this study, we observed that one Rhs immunity protein can protect against more than one toxin. Therefore, the internal expression of RhsCT-I could be important for protection against delivered Rhs toxins in addition to regulating Salmonellagrowth in macrophages. The internal transcripts of rhsCT-I are upregulated in InSPI-2 and within macrophages by the combined action of RpoS, PhoP/Q and H-NS. These are the same regulators that control the expression of the type 6 secretion system known to deliver Rhs effectors in Salmonella. This suggests that expression of rhsCT-I is elevated under conditions that favor delivery, which further supports a role for the internal expression of Rhs proteins in protection against toxin delivery.
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| 7. |
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| 8. |
- Schlegel, Susan, et al.
(författare)
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Bacterial-based membrane protein production
- 2014
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1843:8, s. 1739-1749
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Forskningsöversikt (refereegranskat)abstract
- Escherichia coli is by far the most widely used bacterial host for the production of membrane proteins. Usually, different strains, culture conditions and production regimes are screened for to design the optimal production process. However, these E. coli-based screening approaches often do not result in satisfactory membrane protein production yields. Recently, it has been shown that (i) E. coli strains with strongly improved membrane protein production characteristics can be engineered or selected for, (ii) many membrane proteins can be efficiently produced in E. coli-based cell-free systems, (iii) bacteria other than E. coli can be used for the efficient production of membrane proteins, and, (iv) membrane protein variants that retain functionality but are produced at higher yields than the wild-type protein can be engineered or selected for. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
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| 9. |
- Schlegel, Susan, et al.
(författare)
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De-convoluting the Genetic Adaptations of E-coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields
- 2015
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 10:10, s. 1758-1766
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Tidskriftsartikel (refereegranskat)abstract
- The well-established E. coli protein production strain C41(DE3) was isolated from the T7 RNA polymerase-based BL21(DE3) strain for its ability to produce difficult recombinant proteins, and it acquired multiple mutations during its isolation. Standard allelic replacement and competition experiments were insufficient to de-convolute these mutations. By reconstructing the evolution of C41(DE3) in real time, we identified the time frames when the different mutations occurred, enabling us to link them to particular stress events. Starvation stress imposed by the isolation procedure selected for mutations enhancing nutrient uptake, and protein production stress for mutations weakening the lacUV5 promoter, which governs t7rnap expression. Moreover, recapitulating protein production stress in BL21(DE3) showed that mutations weakening the lacUV5 promoter occur through RecA-dependent recombination with the wild-type lac-promoter and are selected for upon the production of any protein. Thus, the instability of the lacUV5 promoter in BL21(DE3) alleviates protein production stress and can be harnessed to enhance production.
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| 10. |
- Schlegel, Susan, 1981-
(författare)
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From protein production to genome evolution in Escherichia coli
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of my Ph.D. studies was to improve production yields of membrane- and secretory proteins in the widely used E. coli protein production strain BL21(DE3). In this strain expression of the gene encoding the protein of interest is driven by the powerful T7 RNA polymerase (T7 RNAP) whose gene is located on the chromosome and under control of the strong, IPTG-inducible lacUV5 promoter. Unfortunately, the production of many membrane and secretory proteins is 'toxic' to BL21(DE3), resulting in poor growth and low production yields.To understand this ‘toxicity’, the BL21(DE3) derived mutant strains C41(DE3) and C43(DE3) were characterized. Somehow, these strains can efficiently produce many ‘toxic’ membrane and secretory proteins. We showed that mutations weakening the lacUV5 promoter are responsible for this. These mutations result in a slower onset of protein production upon the addition of IPTG, which avoids saturating the Sec-translocon capacity. The Sec-translocon is a protein-conducting channel in the cytoplasmic membrane mediating the biogenesis of membrane proteins and translocation of secretory proteins. Next, we constructed a BL21(DE3)-derivative, Lemo21(DE3), in which the activity of T7 RNAP can be precisely controlled by titrating in its natural inhibitor T7 lysozyme using the rhamnose promoter system. In Lemo21(DE3), the expression level of genes encoding membrane and secretory proteins can be set such that the Sec-translocon capacity is not saturated. This is key to optimizing membrane and secretory protein production yields. Finally, reconstructing the evolution of C41(DE3) from BL21(DE3) in real time showed that during its isolation C41(DE3) had acquired mutations critical for surviving the starvation conditions used, and provided insight in how the mutations in the lacUV5 promoter had occurred.
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