| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Mishra, Yogesh, et al.
(författare)
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Active-site plasticity revealed in the asymmetric dimer of AnPrx6 the 1-Cys peroxiredoxin and molecular chaperone from Anabaena sp. PCC 7120
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Peroxiredoxins (Prxs) are vital regulators of intracellular reactive oxygen species levels in all living organisms. Their activity depends on one or two catalytically active cysteine residues, the peroxidatic Cys (C-P) and, if present, the resolving Cys (C-R). A detailed catalytic cycle has been derived for typical 2-Cys Prxs, however, little is known about the catalytic cycle of 1-Cys Prxs. We have characterized Prx6 from the cyanobacterium Anabaena sp. strain PCC7120 (AnPrx6) and found that in addition to the expected peroxidase activity, AnPrx6 can act as a molecular chaperone in its dimeric state, contrary to other Prxs. The AnPrx6 crystal structure at 2.3 angstrom resolution reveals different active site conformations in each monomer of the asymmetric obligate homo-dimer. Molecular dynamic simulations support the observed structural plasticity. A FSH motif, conserved in 1-Cys Prxs, precedes the active site PxxxTxxCp signature and might contribute to the 1-Cys Prx reaction cycle.
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| 3. |
- Mishra, Yogesh, et al.
(författare)
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Expression, purification, crystallization and preliminary X-ray crystallographic studies of alkyl hydroperoxide reductase (AhpC) from the cyanobacterium Anabaena sp. PCC 7120
- 2011
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Ingår i: Acta Crystallographica. Section F. - : International Union of Crystallography. - 1744-3091 .- 1744-3091. ; 67:10, s. 1203-1206
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Tidskriftsartikel (refereegranskat)abstract
- Alkyl hydroperoxide reductase (AhpC) is a key component of a large family of thiol-specific antioxidant (TSA) proteins distributed among prokaryotes and eukaryotes. AhpC is involved in the detoxification of reactive oxygen species (ROS) and reactive sulfur species (RSS). Sequence analysis of AhpC from the cyanobacterium Anabaena sp. PCC 7120 shows that this protein belongs to the 1-Cys class of peroxiredoxins (Prxs). It has recently been reported that enhanced expression of this protein in Escherichia coli offers tolerance to multiple stresses such as heat, salt, copper, cadmium, pesticides and UV-B. However, the structural features and the mechanism behind this process remain unclear. To provide insights into its biochemical function, AhpC was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. Diffraction data were collected to a maximum d-spacing of 2.5 Å using synchrotron radiation. The crystal belonged to space group P212121, with unit-cell parameters a = 80, b = 102, c = 109.6 Å. The structure of AhpC from Anabaena sp. PCC 7120 was determined by molecular-replacement methods using the human Prx enzyme hORF6 (PDB entry1prx) as the template.
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| 4. |
- Molin, Mikael, 1973, et al.
(författare)
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Protein kinase A controls yeast growth in visible light
- 2020
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Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400–700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection. Results: We found genes involved in HOG pathway signaling, RNA polymerase II transcription, translation, diphthamide modifications of the translational elongation factor eEF2, and the oxidative stress response to be required for light resistance. Reduced nuclear localization of the transcription factor Msn2 and lower glycogen accumulation indicated higher protein kinase A (cAMP-dependent protein kinase, PKA) activity in many light-sensitive gene deletion strains. We therefore used an ectopic fluorescent PKA reporter and mutants with constitutively altered PKA activity to show that repression of PKA is essential for resistance to visible light. Conclusion: We conclude that yeast photobiology is multifaceted and that protein kinase A plays a key role in the ability of cells to grow upon visible light exposure. We propose that visible light impacts on the biology and evolution of many non-photosynthetic organisms and have practical implications for how organisms are studied in the laboratory, with or without illumination.
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| 5. |
- Sharma, Neha, et al.
(författare)
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Fungal-bacterial combinations in plant health under stress : physiological and biochemical characteristics of the filamentous fungus serendipita indica and the actinobacterium zhihengliuella sp. ISTPL4 under in vitro arsenic stress
- 2024
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Ingår i: Microorganisms. - : MDPI. - 2076-2607. ; 12:2
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Tidskriftsartikel (refereegranskat)abstract
- Fungal-bacterial combinations have a significant role in increasing and improving plant health under various stress conditions. Metabolites secreted by fungi and bacteria play an important role in this process. Our study emphasizes the significance of secondary metabolites secreted by the fungus Serendipita indica alone and by an actinobacterium Zhihengliuella sp. ISTPL4 under normal growth conditions and arsenic (As) stress condition. Here, we evaluated the arsenic tolerance ability of S. indica alone and in combination with Z. sp. ISTPL4 under in vitro conditions. The growth of S. indica and Z. sp. ISTPL4 was measured in varying concentrations of arsenic and the effect of arsenic on spore size and morphology of S. indica was determined using confocal microscopy and scanning electron microscopy. The metabolomics study indicated that S. indica alone in normal growth conditions and under As stress released pentadecanoic acid, glycerol tricaprylate, L-proline and cyclo(L-prolyl-L-valine). Similarly, d-Ribose, 2-deoxy-bis(thioheptyl)-dithioacetal were secreted by a combination of S. indica and Z. sp. ISTPL4. Confocal studies revealed that spore size of S. indica decreased by 18% at 1.9 mM and by 15% when in combination with Z. sp. ISTPL4 at a 2.4 mM concentration of As. Arsenic above this concentration resulted in spore degeneration and hyphae fragmentation. Scanning electron microscopy (SEM) results indicated an increased spore size of S. indica in the presence of Z. sp. ISTPL4 (18 ± 0.75 µm) compared to S. indica alone (14 ± 0.24 µm) under normal growth conditions. Our study concluded that the suggested combination of microbial consortium can be used to increase sustainable agriculture by combating biotic as well as abiotic stress. This is because the metabolites released by the microbial combination display antifungal and antibacterial properties. The metabolites, besides evading stress, also confer other survival strategies. Therefore, the choice of consortia and combination partners is important and can help in developing strategies for coping with As stress.
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| 6. |
- Yadav, Sandhya, et al.
(författare)
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Comparison and optimization of protein extraction and two-dimensional gel electrophoresis protocols for liverworts
- 2020
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Ingår i: BMC Research Notes. - : Springer Nature. - 1756-0500. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Objective Liverworts possess historical adaptive strategies for abiotic stresses because they were the first plants that shifted from water to land. Proteomics is a state-of-the-art technique that can capture snapshots of events occurring at the protein level in many organisms. Herein, we highlight the comparison and optimization of an effective protein extraction and precipitation protocol for two-dimensional gel electrophoresis (2-DE) of liverworts. Results We compared three different protein extraction methods, i.e.,1.5 M Tris-HCl (pH 8.8), 50 mM Tris-HCl (pH 7.5), and polyvinylpolypyrrolidone (PVPP) extraction, followed by three precipitation methods, i.e., 80% ethanol, 80% acetone, and 20% tricholoroacetic acid (TCA)-acetone, in a liverwort Dumortiera hirsuta. Among these methods, 50 mM Tris-HCl (pH 7.5) extraction, followed by 20% TCA-acetone precipitation, appeared to be more suitable for 2-DE. Furthermore, we performed modifications during protein washing, re-solubilization in rehydration buffer and isoelectric focusing (IEF). The modifications provided us better results in terms of protein yield, resolution, spot numbers, and intensities for 2-DE gels of D. hirsuta and other two liverworts, i.e., Marchantia paleacea and Plagiochasma appendiculatum. Furthermore, we randomly selected spots from the 2-DE gel of D. hirsuta and identified using mass spectrometry, which confirms the applicability of this protocol for liverworts proteomics.
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