A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T

The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at high salinity by synthesizing and accumulating the compatible solute ectoine. Ectoine levels are highly regulated according to external salt levels but the overall picture of its metabolism and control is not well understood. Apart from its critical role in cell adaptation to halophilic environments, ectoine can be used as a stabilizer for enzymes and as a cell protectant in skin and health care applications and is thus produced annually on a scale of tons in an industrial process using H. elongata as producer strain. This paper presents the complete genome sequence of H. elongata (4 061 296 bp) and includes experiments and analysis identifying and characterizing the entire ectoine metabolism, including a newly discovered pathway for ectoine degradation and its cyclic connection to ectoine synthesis. The degradation of ectoine (doe) proceeds via hydrolysis of ectoine (DoeA) to Nα-acetyl-l-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). In H. elongata, diaminobutyric acid can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation. Genome comparison revealed that the ectoine degradation pathway exists predominantly in non-halophilic bacteria unable to synthesize ectoine. Based on the resulting genetic and biochemical data, a metabolic flux model of ectoine metabolism was derived that can be used to understand the way H. elongata survives under varying salt stresses and that provides a basis for a model-driven improvement of industrial ectoine production

Neutrons describe ectoine effects on water H-bonding and hydration around a soluble protein and a cell membrane

Understanding adaptation to extreme environments remains a challenge of high biotechnological potential for fundamental molecular biology. The cytosol of many microorganisms, isolated from saline environments, reversibly accumulates molar concentrations of the osmolyte ectoine to counterbalance fluctuating external salt concentrations. Although they have been studied extensively by thermodynamic and spectroscopic methods, direct experimental structural data have, so far, been lacking on ectoine-water-protein interactions. In this paper, in vivo deuterium labeling, small angle neutron scattering, neutron membrane diffraction and inelastic scattering are combined with neutron liquids diffraction to characterize the extreme ectoine-containing solvent and its effects on purple membrane of H. salinarum and E. coli maltose binding protein. The data reveal that ectoine is excluded from the hydration layer at the membrane surface and does not affect membrane molecular dynamics, and prove a previous hypothesis that ectoine is excluded from a monolayer of dense hydration water around the soluble protein. Neutron liquids diffraction to atomic resolution shows how ectoine enhances the remarkable properties of H-bonds in water-properties that are essential for the proper organization, stabilization and dynamics of biological structures

Kinetics of the urea–urease clock reaction with urease immobilized in hydrogel beads

Feedback driven by enzyme catalyzed reactions occurs widely in biology and has been well characterized in single celled organisms such as yeast. There are still few examples of robust enzyme oscillators in vitro that might be used to study nonlinear dynamical behavior. One of the simplest is the urea–urease reaction that displays autocatalysis driven by the increase in pH accompanying the production of ammonia. A clock reaction was obtained from low to high pH in batch reactor and bistability and oscillations were reported in a continuous flow rector. However, the oscillations were found to be irreproducible and one contributing factor may be the lack of stability of the enzyme in solution at room temperature. Here, we investigated the effect of immobilizing urease in thiol-poly(ethylene glycol) acrylate (PEGDA) hydrogel beads, prepared using emulsion polymerization, on the urea–urease reaction. The resultant mm-sized beads were found to reproduce the pH clock and, under the conditions employed here, the stability of the enzyme was increased from hours to days

PH wave-front propagation in the urea-urease reaction

The urease-catalyzed hydrolysis of urea displays feedback that results in a switch from acid (pH ∼3) to base (pH ∼9) after a controllable period of time (from 10 to \u3e5000 s). Here we show that the spatially distributed reaction can support pH wave fronts propagating with a speed of the order of 0.1-1 mm min-1. The experimental results were reproduced qualitatively in reaction-diffusion simulations including a Michaelis-Menten expression for the urease reaction with a bell-shaped rate-pH dependence. However, this model fails to predict that at lower enzyme concentrations, the unstirred reaction does not always support fronts when the well-stirred reaction still rapidly switches to high pH. © 2012 by the Biophysical Society

Turing Patterns Inside Cells

Concentration gradients inside cells are involved in key processes such as cell division and morphogenesis. Here we show that a model of the enzymatic step catalized by phosphofructokinase (PFK), a step which is responsible for the appearance of homogeneous oscillations in the glycolytic pathway, displays Turing patterns with an intrinsic length-scale that is smaller than a typical cell size. All the parameter values are fully consistent with classic experiments on glycolytic oscillations and equal diffusion coefficients are assumed for ATP and ADP. We identify the enzyme concentration and the glycolytic flux as the possible regulators of the pattern. To the best of our knowledge, this is the first closed example of Turing pattern formation in a model of a vital step of the cell metabolism, with a built-in mechanism for changing the diffusion length of the reactants, and with parameter values that are compatible with experiments. Turing patterns inside cells could provide a check-point that combines mechanical and biochemical information to trigger events during the cell division process

Diffusive coupling can discriminate between similar reaction mechanisms in an allosteric enzyme system

<p>Abstract</p> <p>Background</p> <p>A central question for the understanding of biological reaction networks is how a particular dynamic behavior, such as bistability or oscillations, is realized at the molecular level. So far this question has been mainly addressed in well-mixed reaction systems which are conveniently described by ordinary differential equations. However, much less is known about how molecular details of a reaction mechanism can affect the dynamics in diffusively coupled systems because the resulting partial differential equations are much more difficult to analyze.</p> <p>Results</p> <p>Motivated by recent experiments we compare two closely related mechanisms for the product activation of allosteric enzymes with respect to their ability to induce different types of reaction-diffusion waves and stationary Turing patterns. The analysis is facilitated by mapping each model to an associated complex Ginzburg-Landau equation. We show that a sequential activation mechanism, as implemented in the model of Monod, Wyman and Changeux (MWC), can generate inward rotating spiral waves which were recently observed as glycolytic activity waves in yeast extracts. In contrast, in the limiting case of a simple Hill activation, the formation of inward propagating waves is suppressed by a Turing instability. The occurrence of this unusual wave dynamics is not related to the magnitude of the enzyme cooperativity (as it is true for the occurrence of oscillations), but to the sensitivity with respect to changes of the activator concentration. Also, the MWC mechanism generates wave patterns that are more stable against long wave length perturbations.</p> <p>Conclusions</p> <p>This analysis demonstrates that amplitude equations, which describe the spatio-temporal dynamics near an instability, represent a valuable tool to investigate the molecular effects of reaction mechanisms on pattern formation in spatially extended systems. Using this approach we have shown that the occurrence of inward rotating spiral waves in glycolysis can be explained in terms of an MWC, but not with a Hill mechanism for the activation of the allosteric enzyme phosphofructokinase. Our results also highlight the importance of enzyme oligomerization for a possible experimental generation of Turing patterns in biological systems.</p

Single-cell analysis reveals individual spore responses to simulated space vacuum

Outer space is a challenging environment for all forms of life, and dormant spores of bacteria have been frequently used to study the survival of terrestrial life in a space journey. Previous work showed that outer space vacuum alone can kill bacterial spores. However, the responses and mechanisms of resistance of individual spores to space vacuum are unclear. Here, we examined spores’ molecular changes under simulated space vacuum (~10−5 Pa) using micro-Raman spectroscopy and found that this vacuum did not cause significant denaturation of spore protein. Then, live-cell microscopy was developed to investigate the temporal events during germination, outgrowth, and growth of individual Bacillus spores. The results showed that after exposure to simulated space vacuum for 10 days, viability of spores of two Bacillus species was reduced up to 35%, but all spores retained their large Ca2 +-dipicolinic acid depot. Some of the killed spores did not germinate, and the remaining germinated but did not proceed to vegetative growth. The vacuum treatment slowed spore germination, and changed average times of all major germination events. In addition, viable vacuum-treated spores exhibited much greater sensitivity than untreated spores to dry heat and hyperosmotic stress. Among spores’ resistance mechanisms to high vacuum, DNA-protective α/β−type small acid-soluble proteins, and non- homologous end joining and base excision repair of DNA played the most important roles, especially against multiple cycles of vacuum treatment. Overall, these results give new insight into individual spore’s responses to space vacuum and provide new techniques for microorganism analysis at the single-cell level

Genomics, evolution, and crystal structure of a new family of bacterial spore kinases

Bacterial spore formation is a complex process of fundamental relevance to biology and human disease. The spore coat structure is complex and poorly understood, and the roles of many of the protein components remain unclear. We describe a new family of spore coat proteins, the bacterial spore kinases (BSKs), and the first crystal structure of a BSK, YtaA (CotI) from Bacillus subtilis. BSKs are widely distributed in spore-forming Bacillus and Clostridium species, and have a dynamic evolutionary history. Sequence and structure analyses indicate that the BSKs are CAKs, a prevalent group of small molecule kinases in bacteria that is distantly related to the eukaryotic protein kinases. YtaA has substantial structural similarity to CAKs, but also displays distinctive features that broaden our understanding of the CAK group. Evolutionary constraint analysis of the protein surfaces indicates that members of the BSK family have distinct clade-conserved patterns in the substrate binding region, and probably bind and phosphorylate distinct targets. Several classes of BSKs have apparently independently lost catalytic activity to become pseudokinases, indicating that the family also has a major noncatalytic function. Proteins 2010. © 2009 Wiley-Liss, Inc

A transposon present in specific strains of Bacillus subtilis negatively affects nutrient- and dodecylamine-induced spore germination

Spore germination shows a large inter-strain variability. Spores of certain Bacillus subtilis strains, including isolates from spoiled food products, exhibit different germination behavior from spores of the well-studied model organism Bacillus subtilis 168, often for unknown reasons. In this study, we analyzed spore germination efficiencies and kinetics of seventeen B. subtilis strains with previously sequenced genomes. A subsequent gene-trait matching analysis revealed a correlation between a slow germination phenotype and the presence of a mobile genetic element, i.e. a Tn1546-like transposon. A detailed investigation of the transposon elements showed an essential role of a specific operon (spoVA(2mob) ) in inhibiting spore germination with nutrients and with the cationic surfactant dodecylamine. Our results indicate that this operon negatively influences release of Ca-DPA by the SpoVA channel and may additionally alter earlier germination events, potentially by affecting proteins in the spore inner membrane. The spoVA(2mob) operon is an important factor that contributes to inter-strain differences in spore germination. Screening for its genomic presence can be applied for identification of spores that exhibit specific properties that impede spore eradication by industrial processes. This article is protected by copyright. All rights reserved

A Negative Feedback Loop That Limits the Ectopic Activation of a Cell Type–Specific Sporulation Sigma Factor of Bacillus subtilis

Two highly similar RNA polymerase sigma subunits, σF and σG, govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σF drives synthesis of σG but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σG while discriminating between σF and σG in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σG (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σG in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σG. CsfB is normally produced in the forespore, under σF control, but sigGN45E mutant cells also expressed csfB and did so in a σG-dependent manner, autonomously from σF. Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σG activity. N45 is invariant in the homologous position of σG orthologues, whereas its functional equivalent in σF proteins, E39, is highly conserved. While CsfB does not bind to wild-type σF, a E39N substitution in σF resulted in efficient binding of CsfB to σF. Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σF in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore-specific sigma factors in B. subtilis