Transcriptional regulation and energetics of alternative respiratory pathways in facultatively anaerobic bacteria

AbstractThe facultatively anaerobic Escherichia coli is able to grow by aerobic and by anaerobic respiration. Despite the large difference in the amount of free energy that could maximally be conserved from aerobic versus anaerobic respiration, the proton potential and Δg′Phos are similar under both conditions. O2 represses anaerobic respiration, and nitrate represses fumarate respiration. By this the terminal reductases of aerobic and anaerobic respiration are expressed in a way to obtain maximal H+e− ratios and ATP yields. The respiratory dehydrogenases, on the other hand, are not synthesized in a way to achieve maximal H+e− ratios. Most of the dehydrogenases of aerobic respiration do not conserve redox enery in a proton gradient whereas the enzymes from anaerobic respiration do so. Thus transcriptional regulation of the respiratory pathways by electron acceptors has multiple effects on cellular energetics. The transcriptional regulation in response to O2 is effected by two transcriptional regulators, ArcA/B (aerobic respiratory control) and FNR (fumarate nitrate reductase regulator). FNR contains an O2-sensitive [4Fe4S]2+ cluster in the sensory domain and is converted to the transcriptional inactive state in the presence of (cytoplasmic) O2

The Cdc42 effectors Ste20, Cla4, and Skm1 down-regulate the expression of genes involved in sterol uptake by a mitogen-activated protein kinase-independent pathway

© 2009 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0).In Saccharomyces cerevisiae, the Rho-type GTPase Cdc42 regulates polarized growth through its effectors, including the p21-activated kinases (PAKs) Ste20, Cla4, and Skm1. Previously, we demonstrated that Ste20 interacts with several proteins involved in sterol synthesis that are crucial for cell polarization. Under anaerobic conditions, sterols cannot be synthesized and need to be imported into cells. Here, we show that Ste20, Cla4, and Skm1 form a complex with Sut1, a transcriptional regulator that promotes sterol uptake. All three PAKs can translocate into the nucleus and down-regulate the expression of genes involved in sterol uptake, including the Sut1 targets AUS1 and DAN1 by a novel mechanism. Consistently, deletion of either STE20, CLA4, or SKM1 results in an increased sterol influx and PAK overexpression inhibits sterol uptake. For Ste20, we demonstrate that the down-regulation of gene expression requires nuclear localization and kinase activity of Ste20. Furthermore, the Ste20-mediated control of expression of sterol uptake genes depends on SUT1 but is independent of a mitogen-activated protein kinase signaling cascade. Together, these observations suggest that PAKs translocate into the nucleus, where they modulate expression of sterol uptake genes via Sut1, thereby controlling sterol homeostasis.Deutsche Forschungsgemeinschaft and the Swiss National Science Foundation

Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors

AbstractThe electron-transport chains of Escherichia coli are composed of many different dehydrogenases and terminal reductases (or oxidases) which are linked by quinones (ubiquinone, menaquinone and demethylmenaquinone). Quinol:cytochrome c oxido-reductase (`bc1 complex') is not present. For various electron acceptors (O2, nitrate) and donors (formate, H2, NADH, glycerol-3-P) isoenzymes are present. The enzymes show great variability in membrane topology and energy conservation. Energy is conserved by conformational proton pumps, or by arrangement of substrate sites on opposite sides of the membrane resulting in charge separation. Depending on the enzymes and isoenzymes used, the H+/e− ratios are between 0 and 4 H+/e− for the overall chain. The expression of the terminal reductases is regulated by electron acceptors. O2 is the preferred electron acceptor and represses the terminal reductases of anaerobic respiration. In anaerobic respiration, nitrate represses other terminal reductases, such as fumarate or DMSO reductases. Energy conservation is maximal with O2 and lowest with fumarate. By this regulation pathways with high ATP or growth yields are favoured. The expression of the dehydrogenases is regulated by the electron acceptors, too. In aerobic growth, non-coupling dehydrogenases are expressed and used preferentially, whereas in fumarate or DMSO respiration coupling dehydrogenases are essential. Coupling and non-coupling isoenzymes are expressed correspondingly. Thus the rationale for expression of the dehydrogenases is not maximal energy yield, but could be maximal flux or growth rates. Nitrate regulation is effected by two-component signal transfer systems with membraneous nitrate/nitrite sensors (NarX, NarQ) and cytoplasmic response regulators (NarL, NarP) which communicate by protein phosphorylation. O2 regulates by a two-component regulatory system consisting of a membraneous sensor (ArcB) and a response regulator (ArcA). ArcA is the major regulator of aerobic metabolism and represses the genes of aerobic metabolism under anaerobic conditions. FNR is a cytoplasmic O2 responsive regulator with a sensory and a regulatory DNA-binding domain. FNR is the regulator of genes required for anaerobic respiration and related pathways. The binding sites of NarL, NarP, ArcA and FNR are characterized for various promoters. Most of the genes are regulated by more than one of the regulators, which can act in any combination and in a positive or negative mode. By this the hierarchical expression of the genes in response to the electron acceptors is achieved. FNR is located in the cytoplasm and contains a 4Fe4S cluster in the sensory domain. The regulatory concentrations of O2 are 1–5 mbar. Under these conditions O2 diffuses to the cytoplasm and is able to react directly with FNR without involvement of other specific enzymes or protein mediators. By oxidation of the FeS cluster, FNR is converted to the inactive state in a reversible process. Reductive activation could be achieved by cellular reductants in the absence of O2. In addition, O2 may cause destruction and loss of the FeS cluster. It is not known whether this process is required for regulation of FNR function

Autonomous Calibration of Single Spin Qubit Operations

Fully autonomous precise control of qubits is crucial for quantum information processing, quantum communication, and quantum sensing applications. It requires minimal human intervention on the ability to model, to predict and to anticipate the quantum dynamics [1,2], as well as to precisely control and calibrate single qubit operations. Here, we demonstrate single qubit autonomous calibrations via closed-loop optimisations of electron spin quantum operations in diamond. The operations are examined by quantum state and process tomographic measurements at room temperature, and their performances against systematic errors are iteratively rectified by an optimal pulse engineering algorithm. We achieve an autonomous calibrated fidelity up to 1.00 on a time scale of minutes for a spin population inversion and up to 0.98 on a time scale of hours for a Hadamard gate within the experimental error of 2%. These results manifest a full potential for versatile quantum nanotechnologies.Comment: 9 pages, 5 figure

Controllable Non-Markovianity for a Spin Qubit in Diamond

We present a flexible scheme to realize non-artificial non-Markovian dynamics of an electronic spin qubit, using a nitrogen-vacancy center in diamond where the inherent nitrogen spin serves as a regulator of the dynamics. By changing the population of the nitrogen spin, we show that we can smoothly tune the non-Markovianity of the electron spin's dynamic. Furthermore, we examine the decoherence dynamics induced by the spin bath to exclude other sources of non-Markovianity. The amount of collected measurement data is kept at a minimum by employing Bayesian data analysis. This allows for a precise quantification of the parameters involved in the description of the dynamics and a prediction of so far unobserved data points.Comment: 12 pages, 9 figure, including supplemental materia

Influence of association state and DNA binding on the O2-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator

The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2

Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase

Transmembrane (TM) signaling is a key process of membrane-bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by TM helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in TM signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable TM homodimer in both the inactive and fumarate-activated state of DcuS. An S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain and on the cytoplasmic side into the α1-helix of PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC contains an LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PASP (α6) domain across the membrane to α1(PASC). Taken together, the results suggest piston-type TM signaling by the TM2 homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PASC and kinase regulation