Fred C. Boogerd

Emergence and its place in nature; a case study of biochemical networks

We will show that there is a strong form of emergence in cell biology. Beginning with C.D. Broad’s classic discussion of emergence, we distinguish two conditions sufficient for emergence. Emergence in biology must be compatible with the thought that all explanations of systemic properties are mechanistic explanations and with their sufficiency. Explanations of systemic properties are always in terms of the properties of the parts within the system. Nonetheless, systemic properties can still be emergent. If the properties of the components within the system cannot be predicted, even in principle, from the behavior of the system’s parts within simpler wholes then there also will be systemic properties which cannot be predicted, even in principle, on basis of the behavior of these parts. We show in an explicit case study drawn from molecular cell physiology that biochemical networks display this kind of emergence, even though they deploy only mechanistic explanations. This illustrates emergence and its place in nature.

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

SUMMARY We present a comprehensive overview of the hierarchical network of intracellular processes revolving around central nitrogen metabolism in Escherichia coli. The hierarchy intertwines transport, metabolism, signaling leading to posttranslational modification, and transcription. The protein components of the network include an ammonium transporter (AmtB), a glutamine transporter (GlnHPQ), two ammonium assimilation pathways (glutamine synthetase [GS]-glutamate synthase [glutamine 2-oxoglutarate amidotransferase {GOGAT}] and glutamate dehydrogenase [GDH]), the two bifunctional enzymes adenylyl transferase/adenylyl-removing enzyme (ATase) and uridylyl transferase/uridylyl-removing enzyme (UTase), the two trimeric signal transduction proteins (GlnB and GlnK), the two-component regulatory system composed of the histidine protein kinase nitrogen regulator II (NRII) and the response nitrogen regulator I (NRI), three global transcriptional regulators called nitrogen assimilation control (Nac) protein, leucine-responsive regulatory protein (Lrp), and cyclic AMP (cAMP) receptor protein (Crp), the glutaminases, and the nitrogen-phosphotransferase system. First, the structural and molecular knowledge on these proteins is reviewed. Thereafter, the activities of the components as they engage together in transport, metabolism, signal transduction, and transcription and their regulation are discussed. Next, old and new molecular data and physiological data are put into a common perspective on integral cellular functioning, especially with the aim of resolving counterintuitive or paradoxical processes featured in nitrogen assimilation. Finally, we articulate what still remains to be discovered and what general lessons can be learned from the vast amounts of data that are available now.

Electron transport to nitrous oxide in Paracoccus denitrificans.

Many bacteria can use nitrogenous oxides, instead of oxygen, as terminal electron acceptor during respiration [ 11. Puracoccus denitrijkans is capable of growing anaerobically in the presence of nitrate, nitrite or nitrous oxide. When nitrate is the electron acceptor growth is accompanied by the evolution of nitrogen and nitrous oxide [2]. Cells grown anaerobically with nitrate as electron acceptor contain flavin, UQ-10, b-, c-, and o-type cytochromes, a membrane-bound nitrate-reductase and a soluble two-haem (cand dtype) nitrite-reductase [3,4]. There are strong indications that b-type cytochromes are involved in electron transport to nitrate and c-type cytochromes in electron transport to nitrite [5,6]. The occurrence of obligate intermediates, like nitrous oxide, in the reduction of nitrite to nitrogen gas is a point of controversy [7-lo]. Therefore we started a more profound study of the role of nitrous oxide in denitrification by Pa. denitrificans. The results indicate that nitrous oxide could be an obligate intermediate in the reduction of nitrite to nitrogen gas and that ctype cytochrome(s) are involved in electron transport to nitrous oxide.

The multifarious short‐term regulation of ammonium assimilation of Escherichia coli: dissection using an in silico replica

Ammonium assimilation in Escherichia coli is regulated through multiple mechanisms (metabolic, signal transduction leading to covalent modification, transcription, and translation), which (in‐)directly affect the activities of its two ammonium‐assimilating enzymes, i.e. glutamine synthetase (GS) and glutamate dehydrogenase (GDH). Much is known about the kinetic properties of the components of the regulatory network that these enzymes are part of, but the ways in which, and the extents to which the network leads to subtle and quasi‐intelligent regulation are unappreciated. To determine whether our present knowledge of the interactions between and the kinetic properties of the components of this network is complete − to the extent that when integrated in a kinetic model it suffices to calculate observed physiological behaviour − we now construct a kinetic model of this network, based on all of the kinetic data on the components that is available in the literature. We use this model to analyse regulation of ammonium assimilation at various carbon statuses for cells that have adapted to low and high ammonium concentrations. We show how a sudden increase in ammonium availability brings about a rapid redirection of the ammonium assimilation flux from GS/glutamate synthase (GOGAT) to GDH. The extent of redistribution depends on the nitrogen and carbon status of the cell. We develop a method to quantify the relative importance of the various regulators in the network. We find the importance is shared among regulators. We confirm that the adenylylation state of GS is the major regulator but that a total of 40% of the regulation is mediated by ADP (22%), glutamate (10%), glutamine (7%) and ATP (1%). The total steady‐state ammonium assimilation flux is remarkably robust against changes in the ammonium concentration, but the fluxes through GS and GDH are completely nonrobust. Gene expression of GOGAT above a threshold value makes expression of GS under ammonium‐limited conditions, and of GDH under glucose‐limited conditions, sufficient for ammonium assimilation.

Dissimilatory nitrate uptake in Paracoccus denitrificans via a Δ\̃gmH+-dependent system and a nitrate-nitrite antiport system

Abstract Respiration-driven proton translocation has been studied with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing H2 during reduction of O2, NO−3, NO−2 or N2O. A simplified scheme of anaerobic electron transport and associated proton translocation is shown that is consistent with the measured H + oxidant ratios . Furthermore, the kinetics and energetics of NO−3 uptake in whole cells of P. denitrificans were studied. For this purpose, we measured H2 consumption or N2O production after addition of NO−3 to a cell suspension, which indirectly gave information about uptake (and reduction) of NO−3. It was found that a lag phase in H2 consumption or N2O production appeared whenever the membrane potential was dissipated by addition of thiocyanate, carbonyl cyanide m-chlorophenylhydrazone or triphenyl-methylphosphonium bromide. However, these lag phases were not observed when NO−2 was present at the moment of introduction of NO−3. On the basis of these findings we conclude that there are two uptake systems for NO−3. One system is dependent on the proton-motive force and is probably used for initiation of NO−3 uptake. The other is an NO − 3 NO − 2 antiport and its function is to take over NO−3 uptake from the first system.

AmtB-mediated NH3 transport in prokaryotes must be active and as a consequence regulation of transport by GlnK is mandatory to limit futile cycling of NH4+/NH3

The nature of the ammonium import into prokaryotes has been controversial. A systems biological approach makes us hypothesize that AmtB‐mediated import must be active for intracellular NH 4 + concentrations to sustain growth. Revisiting experimental evidence, we find the permeability assays reporting passive NH3 import inconclusive. As an inevitable consequence of the proposed NH 4 + transport, outward permeation of NH3 constitutes a futile cycle. We hypothesize that the regulatory protein GlnK is required to fine‐tune the active transport of ammonium in order to limit futile cycling whilst enabling an intracellular ammonium level sufficient for the cells nitrogen requirements.

Energetic aspects of growth of Paracoccus denitrificans: oxygen-limitation and shift from anaerobic nitrate-limination to aerobic succinate-limitation

Abstract1. Growth yields and efficiency of energy conservation were the same for aerobic succinate-limited and oxygen-limited cells of Paracoccus denitrificans. 2. A shift from anaerobic nitrate-limitation to aerobic succinatelimitation showed that before and after the shift cells grew with the same capacity of energy conservation. 3. Respiration-driven proton translocation showed the presence of H+-translocating sites 1 and 2, which translocate respectively 2–3 and 4 protons per 2 electrons in oxygen-, anaerobic nitrate-and aerobic succinate-limited cells. 4. Cytochrome spectra and flash-photolysis spectra of oxygen- and nitrate-limited cells gave evidence for the presence of an alternative oxidase, cytochrome a1, never before recognized in Paracoccus denitrificans. 5. Only a-type cytochromes liganded with CO could be flash-photolysed. No evidence for a functional cytochrome o was found in photolysis experiments. 6. Fast oxidation, before photolysis, of the bc-pool after introduction of oxygen in a CO-liganded sample at-20° to-30° C, indicated the presence of a cytochrome oxidase other than cytochrome a1 with a very high affinity for oxygen and a low affinity for CO. 7. In photochemical action spectra, light released CO-inhibition of respiration, but the release was independent of the wavelength used (560–610 nm).

Why in vivo may not equal in vitro – new effectors revealed by measurement of enzymatic activities under the same in vivo‐like assay conditions

Does the understanding of the dynamics of biochemical networks in vivo, in terms of the properties of their components determined in vitro, require the latter to be determined all under the same conditions? An in vivo‐like assay medium for enzyme activity determination was designed based on the concentrations of the major ionic constituents of the Escherichia coli cytosol: K+, Na+, Mg2+, phosphate, glutamate, sulfate and Cl−. The maximum capacities (Vmax) of the extracted enzymes of two pathways were determined using both this in vivo‐like assay medium and the assay medium specific for each enzyme. The enzyme activities differed between the two assay conditions. Most of the differences could be attributed to unsuspected, pleiotropic effects of K+ and phosphate. K+ activated some enzymes (aldolase, enolase and glutamate dehydrogenase) and inhibited others (phosphoglucose isomerase, phosphofructokinase, triosephosphate isomerase, glyceraldehyde 3‐phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase), whereas phosphate inhibited all glycolytic enzymes and glutamine synthetase but only activated glutamine 2‐oxoglutarate amidotransferase. Neither a high glutamate concentration, nor macromolecular crowding affected the glycolytic or nitrogen assimilation enzymes, other than through the product inhibition of glutamate dehydrogenase by glutamate. This strategy of assessing all pathway enzymes kinetically under the same conditions may be necessary to avoid inadvertent differences between in vivo and in vitro biochemistry. It may also serve to reveal otherwise unnoticed pleiotropic regulation, such as that demonstrated in the present study by K+ and phosphate.

Emergence of the silicon human and network targeting drugs

The development of disease may be characterized as a pathological shift of homeostasis; the main goal of contemporary drug treatment is, therefore, to return the pathological homeostasis back to the normal physiological range. From the view point of systems biology, homeostasis emerges from the interactions within the network of biomolecules (e.g. DNA, mRNA, proteins), and, hence, understanding how drugs impact upon the entire network should improve their efficacy at returning the network (body) to physiological homeostasis. Large, mechanism-based computer models, such as the anticipated human whole body models (silicon or virtual human), may help in the development of such network-targeting drugs. Using the philosophical concept of weak and strong emergence, we shall here take a more general look at the paradigm of network-targeting drugs, and propose our approaches to scale the strength of strong emergence. We apply these approaches to several biological examples and demonstrate their utility to reveal principles of bio-modeling. We discuss this in the perspective of building the silicon human.

The bioenergetics of denitrification.

In anaerobically grown Paracoccus denitrificans the dissimilatory nitrate reductase is linked to the respiratory chain at the level of cytochromes b. Electron transport to nitrite and nitrous oxide involves c-type cytochromes. During electron transport from NADH to nitrate one phosphorylation site is passed, whereas two sites are passed during electron transport from NADH to oxygen, nitrite and nitrous oxide. The presentation of a respiratory chain as a linear array of electron carriers gives a misleading picture of the efficiency of energy conservation since the location of the reductases is not taken into account. For the reduction of nitrite and nitrous oxide, protons are utilized from the periplasmic space, whereas for the reduction of oxygen and nitrate, protons are utilized from the cytoplasmic side of the inner membrane. Evidence for two transport systems for nitrate was obtained. One is driven by the proton motive force; this system is used to initiate nitrate reduction. The second system is a nitrate-nitrite antiport system. A scheme for proton translocation and electron transport to nitrate, nitrite, nitrous oxide and oxygen is presented. The number of charges translocated across the membrane during flow of two electrons from NADH is the same for all nitrogenous oxides and is 67-71% of that during electron transfer to oxygen via cytochrome o. These findings are in accordance with growth yield studies. YMAX electron values determined in chemostat cultures for growth with various substrates and hydrogen acceptors are proportional to the number of charges translocated to these hydrogen acceptors during electron transport.