Abstract:Iron is essential for all bacteria. In most bacteria, intracellular iron homeostasis is tightly regulated by the ferric uptake regulator Fur. However, how Fur activates the iron-uptake system during iron deficiency is not fully elucidated. In this study, we found that YdiV, the flagella gene inhibitor, is involved in iron homeostasis in Escherichia coli. Iron deficiency triggers overexpression of YdiV. High levels of YdiV then transforms Fur into a novel form which does not bind DNA in a peptidyl-prolyl cis-tr… Show more
“…Using both the classic Pb(Ac) 2 detection test and a fluorescent-based probe WSP5 (Zhang et al, 2020), we confirmed that, the production of H 2 S in cbl deletion strain was significantly lower than that of WT in M9 medium (Figs. 3A and 3B).…”
Section: Cbl Affects the Generation Of H 2 Ssupporting
confidence: 56%
“…For the EMSA, DNA probe was incubated with Cbl protein samples in reaction buffer (10 mM Tris–HCl, one mM MgCl 2 , one mM DTT, 40 mM KCl, 0.1 mg/mL BSA, 5% (w/v) glycerol) at 37 °C for 30 min. After the samples were separated using a 6% native acrylamide gel ( Zhang et al, 2020 ), the gel was then exposed to a phosphorscreen and visualized on Typhoon FLA 9500.…”
Endogenous hydrogen sulfide (H2S) is generated in many metabolism pathways, and has been recognized as a second messenger against antibiotics and reactive oxygen species (ROS). In Aeromonas veronii, Small Protein B (SmpB) plays an important role in resisting stress. The absence of smpB could trigger sulfate assimilation pathway to adapt the nutrient deficiency, of which was mediated by up-regulation of cbl and cys genes and followed with enhancing H2S production. To figure out the mutual regulations of cbl and cys genes, a series of experiments were performed. Compared with the wild type, cysH was down-regulated significantly in cbl deletion by qRT-PCR. The fluorescence analysis further manifested that Cbl had a positive regulatory effect on the promoter of cysJIH. Bacterial one-hybrid analysis and electrophoretic mobility shift assay (EMSA) verified that Cbl bound with the promoter of cysJIH. Collectively, the tolerance to adversity could be maintained by the production of H2S when SmpB was malfunctioned, of which the activity of cysJIH promoter was positively regulated by upstream Cbl protein. The outcomes also suggested the enormous potentials of Aeromonas veronii in environmental adaptability.
“…Using both the classic Pb(Ac) 2 detection test and a fluorescent-based probe WSP5 (Zhang et al, 2020), we confirmed that, the production of H 2 S in cbl deletion strain was significantly lower than that of WT in M9 medium (Figs. 3A and 3B).…”
Section: Cbl Affects the Generation Of H 2 Ssupporting
confidence: 56%
“…For the EMSA, DNA probe was incubated with Cbl protein samples in reaction buffer (10 mM Tris–HCl, one mM MgCl 2 , one mM DTT, 40 mM KCl, 0.1 mg/mL BSA, 5% (w/v) glycerol) at 37 °C for 30 min. After the samples were separated using a 6% native acrylamide gel ( Zhang et al, 2020 ), the gel was then exposed to a phosphorscreen and visualized on Typhoon FLA 9500.…”
Endogenous hydrogen sulfide (H2S) is generated in many metabolism pathways, and has been recognized as a second messenger against antibiotics and reactive oxygen species (ROS). In Aeromonas veronii, Small Protein B (SmpB) plays an important role in resisting stress. The absence of smpB could trigger sulfate assimilation pathway to adapt the nutrient deficiency, of which was mediated by up-regulation of cbl and cys genes and followed with enhancing H2S production. To figure out the mutual regulations of cbl and cys genes, a series of experiments were performed. Compared with the wild type, cysH was down-regulated significantly in cbl deletion by qRT-PCR. The fluorescence analysis further manifested that Cbl had a positive regulatory effect on the promoter of cysJIH. Bacterial one-hybrid analysis and electrophoretic mobility shift assay (EMSA) verified that Cbl bound with the promoter of cysJIH. Collectively, the tolerance to adversity could be maintained by the production of H2S when SmpB was malfunctioned, of which the activity of cysJIH promoter was positively regulated by upstream Cbl protein. The outcomes also suggested the enormous potentials of Aeromonas veronii in environmental adaptability.
“…The mixture was heated at 95°C for 10 min and slowly cooled to room temperature. About 15 μg of extracted protein in binding buffer (pH 7.5, 10 mM TrisHCl, 1 mM MgCl 2 , 40 mM KCl, 0.1 mg/ml BSA, 5% glycerol) was incubated with 200 fmol of FAM-labeled probes at room temperature for 15 min [ 62 , 63 ]. An antibody for His (1.5 μg) was added to the binding reaction in the supershift experiment.…”
The regulation of glycometabolism homeostasis is vital to maintain health and development of animal and humans; however, the molecular mechanisms by which organisms regulate the glucose metabolism homeostasis from a feeding state switching to a non-feeding state are not fully understood. Using the holometabolous lepidopteran insect Helicoverpa armigera, cotton bollworm, as a model, we revealed that the steroid hormone 20-hydroxyecdysone (20E) upregulated the expression of transcription factor Krüppel-like factor (identified as Klf15) to promote macroautophagy/autophagy, apoptosis and gluconeogenesis during metamorphosis. 20E via its nuclear receptor EcR upregulated Klf15 transcription in the fat body during metamorphosis. Knockdown of Klf15 using RNA interference delayed pupation and repressed autophagy and apoptosis of larval fat body during metamorphosis. KLF15 promoted autophagic flux and transiting to apoptosis. KLF15 bound to the KLF binding site (KLF bs) in the promoter of Atg8 (autophagy-related gene 8/LC3) to upregulate Atg8 expression. Knockdown Atg8 reduced free fatty acids (FFAs), glycerol, free amino acids (FAAs) and glucose levels. However, knockdown of Klf15 accumulated FFAs, glycerol, and FAAs. Glycolysis was switched to gluconeogenesis, trehalose and glycogen synthesis were changed to degradation during metamorphosis, which were accompanied by the variation of the related genes expression. KLF15 upregulated phosphoenolpyruvate carboxykinase (Pepck) expression by binding to KLF bs in the Pepck promoter for gluconeogenesis, which utilised FFAs, glycerol, and FAAs directly or indirectly to increase glucose in the hemolymph. Taken together, 20E via KLF15 integrated autophagy and gluconeogenesis by promoting autophagy-related and gluconeogenesis-related genes expression.
“…These large-amplitude and long time scale motions are certainly characteristic of multidomain transcriptional regulators [126,127]. In many cases, they are coupled or obscured by changes in the oligomerization state of the protein that is derived from intermolecular interactions between the sensory domains or even the DNA-binding domains [128][129][130]. Overall, when extensive conformational changes occur upon inducer binding, it is still a challenge to obtain direct experimental information about dynamic changes.…”
The different niches through which bacteria move during their life cycle require a fast response to the many environmental queues they encounter. The sensing of these stimuli and their correct response is driven primarily by transcriptional regulators. This kind of protein is involved in sensing a wide array of chemical species, a process that ultimately leads to the regulation of gene transcription. The allosteric-coupling mechanism of sensing and regulation is a central aspect of biological systems and has become an important field of research during the last decades. In this review, we summarize the state-of-the-art techniques applied to unravel these complex mechanisms. We introduce a roadmap that may serve for experimental design, depending on the answers we seek and the initial information we have about the system of study. We also provide information on databases containing available structural information on each family of transcriptional regulators. Finally, we discuss the recent results of research about the allosteric mechanisms of sensing and regulation involving many transcriptional regulators of interest, highlighting multipronged strategies and novel experimental techniques. The aim of the experiments discussed here was to provide a better understanding at a molecular level of how bacteria adapt to the different environmental threats they face.
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