Region C of the Escherichia coli heat shock sigma factor RpoH (σ32) contains a turnover element for proteolysis by the FtsH protease

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor σ32 (RpoH). At physiological temperatures, RpoH is rapidly degraded by chaperone-mediated FtsH-dependent proteolysis. Several RpoH residues critical for degradation are located in the highly conserved region 2.1. However, additional residues were predicted to be involved in this process. We introduced mutations in region C of RpoH and found that a double mutation (A131E, K134V) significantly stabilized RpoH against degradation by the FtsH protease. Single-point mutations at these positions only showed a slight effect on RpoH stability. Both double and single amino acid substitutions did not impair sigma factor activity as demonstrated by a groE-lacZ reporter gene fusion, Western blot analysis of heat shock gene expression and increased heat tolerance in the presence of these proteins. Combined mutations in regions 2.1 and C further stabilized RpoH. We also demonstrate that an RpoH fragment composed of residues 37-147 (including regions 2.1 and C) is degraded in an FtsH-dependent manner. We conclude that in addition to the previously described turnover element in region 2.1, a previously postulated second region important for proteolysis of RpoH by FtsH lies in region C of the sigma facto

Lifetime vs. rate capability: Understanding the role of FEC and VC in high-energy Li-ion batteries with nano-silicon anodes

Fluoroethylene carbonate (FEC) and vinylene carbonate (VC) are the most frequently used electrolyte components to enhance the lifetime of anode materials in Li-ion batteries, but for silicon it is still ambiguous when FEC or VC is more beneficial. Herein, a nanostructured silicon/carbon anode derived from low-cost HSiCl3 is tailored by the rational choice of the electrolyte component, to obtain an anode material outperforming current complex silicon structures. We demonstrate highly reversible areal capacities of up to 5 mA h/cm2 at 4.4 mg/cm2 mass loading, a specific capacity of 1280 mA h/gElectrode, a capacity retention of 81% after 500 deep-discharge cycles versus lithium metal and successful full-cell tests with high-voltage cathodes meeting the requirements for real application. Electrochemical impedance spectroscopy and post-mortem investigation provide new insights in tailoring the interfacial properties of silicon-based anodes for high performance anode materials based on an alloying mechanism with large volume changes. The role of fluorine in the FEC-derived interfacial layer is discussed in comparison with the VC-derived layer and possible degradation mechanisms are proposed. We believe that this study gives a valuable understanding and provides new strategies on the facile use of additives for highly reversible silicon anodes in Li-ion batteries.Fil: Jaumann, Tony. Ifw Dresden; AlemaniaFil: Balach, Juan Manuel. Ifw Dresden; AlemaniaFil: Langklotz, Ulrike. Technische Universität Dresden; AlemaniaFil: Sauchuk, Viktar. Fraunhofer Institute for Ceramic Materials and Systems; AlemaniaFil: Fritsch, Marco. Fraunhofer Institute for Ceramic Materials and Systems; AlemaniaFil: Michaelis, Alexander. Technische Universität Dresden; AlemaniaFil: Teltevskij, Valerij. Leibniz Institute for Solid State and Materials Research; AlemaniaFil: Mikhailova, Daria. Leibniz Institute for Solid State and Materials Research; AlemaniaFil: Oswald, Steffen. Leibniz Institute for Solid State and Materials Research; AlemaniaFil: Klose, Markus. Leibniz Institute for Solid State and Materials Research; Alemania. Technische Universität Dresden; AlemaniaFil: Stephani, Guenter. Branch Lab Dresden. Fraunhofer Institute for Manufacturing Technology and Advanced Materials; ArgentinaFil: Hauser, Ralf. Branch Lab Dresden. Fraunhofer Institute for Manufacturing Technology and Advanced Materials; ArgentinaFil: Eckert, Jürgen. Technische Universität Dresden; Alemania. Leibniz Institute for Solid State and Materials Research; AlemaniaFil: Giebeler, Lars. Leibniz Institute for Solid State and Materials Research; Alemania. Technische Universität Dresden; Alemani

SEI-component formation on sub 5 nm sized silicon nanoparticles in Li-ion batteries: The role of electrode preparation, FEC addition and binders

Silicon is a promising negative electrode for secondary lithium-based batteries, but the electrochemical reversibility of particularly nanostructured silicon electrodes drastically depends on their interfacial characteristics, commonly known as the solid electrolyte interface (SEI). The beneficial origin of certain electrolyte additives or different binders is still discussed controversially owing to the challenging peculiarities of interfacial post-mortem investigations of electrodes. In this work, we address the common difficulties of SEI investigations of porous silicon/carbon nanostructures and study the addition of a fluoroethylene carbonate (FEC) as a stabilizing additive as well as the use of two different binders, carboxymethyl cellulose/styrene-butadiene rubber (CMC/SBR) and polyacrylic acid (PAA), for the SEI formation. The electrode is composed of silicon nanocrystallites below 5 nm diameter allowing a detailed investigation of interfacial characteristics of silicon owing to the high surface area. We first performed galvanostatic long-term cycling (400 times) and carried out comprehensive ex situ characterization of the cycled nanocrystalline silicon electrodes with XRD, EDXS, TEM and XPS. We modified the preparation of the electrode for post-mortem characterization to distinguish between electrolyte components and the actual SEI. The impact of the FEC additive and two different binders on the interfacial layer is studied and the occurrence of diverse compounds, in particular LiF, Li2O and phosphates, is discussed. These results help to understand general issues in SEI formation and to pave the way for the development of advanced electrolytes allowing for a long-term performance of nanostructured Si-based electrodes

In situ Raman spectroscopy on silicon nanowire anodes integrated in lithium ion batteries

Rapid decay of silicon anodes during lithiation poses a significant challenge in application of silicon as an anode material in lithium ion batteries. In situ Raman spectroscopy is a powerful method to study the relationship between structural and electrochemical data during electrode cycling and to allow the observation of amorphous as well as liquid and transient species in a battery cell. Herein, we present in situ Raman spectroscopy on high capacity electrode using uncoated and carbon-coated silicon nanowires during first lithiation and delithiation cycle in an optimized lithium ion battery setup and complement the results with operando X-ray reflection diffraction measurements. During lithiation, we were able to detect a new Raman signal at 1859 cm−1 especially on uncoated silicon nanowires. The detailed in situ Raman measurement of the first lithiation/delithiation cycle allowed to differentiate between morphology changes of the electrode as well as interphase formation from electrolyte components

Lifetime vs. rate capability: Understanding the role of FEC and VC in high-energy Li-ion batteries with nano-silicon anodes

Fluoroethylene carbonate (FEC) and vinylene carbonate (VC) are the most frequently used electrolyte components to enhance the lifetime of anode materials in Li-ion batteries, but for silicon it is still ambiguous when FEC or VC is more beneficial. Herein, a nanostructured silicon/carbon anode derived from low-cost HSiCl3 is tailored by the rational choice of the electrolyte component, to obtain an anode material outperforming current complex silicon structures. We demonstrate highly reversible areal capacities of up to 5 mA h/cm2 at 4.4 mg/cm2 mass loading, a specific capacity of 1280 mA h/gElectrode, a capacity retention of 81% after 500 deep-discharge cycles versus lithium metal and successful full-cell tests with high-voltage cathodes meeting the requirements for real application. Electrochemical impedance spectroscopy and post-mortem investigation provide new insights in tailoring the interfacial properties of silicon-based anodes for high performance anode materials based on an alloying mechanism with large volume changes. The role of fluorine in the FEC-derived interfacial layer is discussed in comparison with the VC-derived layer and possible degradation mechanisms are proposed. We believe that this study gives a valuable understanding and provides new strategies on the facile use of additives for highly reversible silicon anodes in Li-ion batteries

Sub-cellular location of FtsH proteases in the cyanobacterium Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid membranes

Marie Curie Initial Training Networks. Grant Number: FP7-PEOPLE-ITN-2008 Biotechnology and Biological Sciences Research Council. Grant Numbers: BB/G021856, BB/F020554/

Regulation of the Escherichia coli HipBA Toxin-Antitoxin System by Proteolysis

Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon- background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence

A Complete Pathway Model for Lipid A Biosynthesis in Escherichia coli.

Lipid A is a highly conserved component of lipopolysaccharide (LPS), itself a major component of the outer membrane of Gram-negative bacteria. Lipid A is essential to cells and elicits a strong immune response from humans and other animals. We developed a quantitative model of the nine enzyme-catalyzed steps of Escherichia coli lipid A biosynthesis, drawing parameters from the experimental literature. This model accounts for biosynthesis regulation, which occurs through regulated degradation of the LpxC and WaaA (also called KdtA) enzymes. The LpxC degradation signal appears to arise from the lipid A disaccharide concentration, which we deduced from prior results, model results, and new LpxK overexpression results. The model agrees reasonably well with many experimental findings, including the lipid A production rate, the behaviors of mutants with defective LpxA enzymes, correlations between LpxC half-lives and cell generation times, and the effects of LpxK overexpression on LpxC concentrations. Its predictions also differ from some experimental results, which suggest modifications to the current understanding of the lipid A pathway, such as the possibility that LpxD can replace LpxA and that there may be metabolic channeling between LpxH and LpxB. The model shows that WaaA regulation may serve to regulate the lipid A production rate when the 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) concentration is low and/or to control the number of KDO residues that get attached to lipid A. Computation of flux control coefficients showed that LpxC is the rate-limiting enzyme if pathway regulation is ignored, but that LpxK is the rate-limiting enzyme if pathway regulation is present, as it is in real cells. Control also shifts to other enzymes if the pathway substrate concentrations are not in excess. Based on these results, we suggest that LpxK may be a much better drug target than LpxC, which has been pursued most often

A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis

Penicillin and related antibiotics disrupt cell wall synthesis to induce bacteriolysis. Lysis in response to these drugs requires the activity of cell wall hydrolases called autolysins, but how penicillins misactivate these deadly enzymes has long remained unclear. Here, we show that alterations in surface polymers called teichoic acids (TAs) play a key role in penicillin-induced lysis of the Gram-positive pathogen Streptococcus pneumoniae (Sp). We find that during exponential growth, Sp cells primarily produce lipid-anchored TAs called lipoteichoic acids (LTAs) that bind and sequester the major autolysin LytA. However, penicillin-treatment or prolonged stationary phase growth triggers the degradation of a key LTA synthase, causing a switch to the production of wall-anchored TAs (WTAs). This change allows LytA to associate with and degrade its cell wall substrate, thus promoting osmotic lysis. Similar changes in surface polymer assembly may underlie the mechanism of antibiotic- and/or growth phase-induced lysis for other important Gram-positive pathogens

Substrate diversity and substrate recognition of the bacterial FtsH and Lon proteases

Durch regulierte Proteolyse kann das zelluläre Proteom schnell und effektiv an die jeweiligen Umweltbedingungen angepasst werden. In Bakterien wird der Energieabhängige Abbau von Proteinen durch die spezifische Substraterkennung von $AAA^{+}$-Proteasen ($\bf A$TPases $\bf a$ssociated with various cellular $\bf a$ctivities) ermöglicht. Während die Protease FtsH in der inneren Membran verankert ist, handelt es sich bei Lon um eine cytoplasmatische Protease. FtsH kontrolliert in $\textit {Escherichia coli}$ unter anderem die Hitzeschockantwort und die Biosynthese von Lipopolysacchariden durch Degradation der Proteine RpoH und LpxC. Im Rahmen dieser Arbeit wurde (i) die Substraterkennung von RpoH durch FtsH, (ii) die Konservierung der Regulation der LPS-Synthese über Proteolyse in verschiedenen Gram-negativen Bakterien sowie (iii) die Substratvielfalt von FtsH analysiert. Weiterhin konnte gezeigt werden, dass der Replikationsinhibitor CspD in $\textit {E. coli}$ über Lon-abhängige Proteolyse reguliert wird.Regulated proteolysis is a powerful mechanism to rapidly adjust the cellular proteome to changing environmental conditions. In bacteria the energy-dependent degradation of proteins is mediated by the specific substrate recognition of $AAA^{+}$ proteases ($\bf A$TPases $\bf a$ssociated with various cellular $\bf a$ctivities). While the protease FtsH is anchored to the inner membrane, Lon is a cytoplasmic protease. In Escherichia coli, FtsH e. g. controls the heat shock response and the biosynthesis of lipopolysaccharides (LPS) through degradation of its substrates RpoH and LpxC. In this work (i) the substrate recognition of RpoH by FtsH, (ii) the conservation of regulation of LPS biosynthesis by proteolysis in different Gram-negative species and (iii) the substrate diversity of the FtsH protease were analyzed. Furthermore it was demonstrated that the replication inhibitor CspD is a substrate of the Lon protease in $\it {E. coli}$