Omics data for sampling thermodynamically feasible kinetic models.

Kinetic models are key to understanding and predicting the dynamic behaviour of metabolic systems. Traditional models require kinetic parameters which are not always available and are often estimated in vitro. Ensemble models overcome this challenge by sampling thermodynamically feasible models around a measured reference point. However, it is unclear if the convenient distributions used to generate the ensemble produce a natural distribution of model parameters and hence if the model predictions are reasonable. In this paper, we produced a detailed kinetic model for the central carbon metabolism of Escherichia coli. The model consists of 82 reactions (including 13 reactions with allosteric regulation) and 79 metabolites. To sample the model, we used metabolomic and fluxomic data from a single steady-state time point for E. coli K-12 MG1655 growing on glucose minimal M9 medium (average sampling time for 1000 models: 11.21 ± 0.14 min). Afterwards, in order to examine whether our sampled models are biologically sound, we calculated Km, Vmax and kcat for the reactions and compared them to previously published values. Finally, we used metabolic control analysis to identify enzymes with high control over the fluxes in the central carbon metabolism. Our analyses demonstrate that our platform samples thermodynamically feasible kinetic models, which are in agreement with previously published experimental results and can be used to investigate metabolic control patterns within cells. This renders it a valuable tool for the study of cellular metabolism and the design of metabolic pathways.

GRASP: a computational platform for building kinetic models of cellular metabolism.

Summary: Kinetic models of metabolism are crucial to understand the inner workings of cell metabolism. By taking into account enzyme regulation, detailed kinetic models can provide accurate predictions of metabolic fluxes. Comprehensive consideration of kinetic regulation requires highly parameterized non-linear models, which are challenging to build and fit using available modelling tools. Here, we present a computational package implementing the GRASP framework for building detailed kinetic models of cellular metabolism. By defining the mechanisms of enzyme regulation and a reference state described by reaction fluxes and their corresponding Gibbs free energy ranges, GRASP can efficiently sample an arbitrarily large population of thermodynamically feasible kinetic models. If additional experimental data are available (fluxes, enzyme and metabolite concentrations), these can be integrated to generate models that closely reproduce these observations using an approximate Bayesian computation fitting framework. Within the same framework, model selection tasks can be readily performed. Availability and implementation: GRASP is implemented as an open-source package in the MATLAB environment. The software runs in Windows, macOS and Linux, is documented (graspk.readthedocs.io) and unit-tested. GRASP is freely available at github.com/biosustain/GRASP. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Molecular Insights into Bacteriophage Evolution toward Its Host.

Bacteriophages (phages), viruses that infect bacteria, are considered to be highly host-specific. To add to the knowledge about the evolution and development of bacteriophage speciation toward its host, we conducted a 21-day experiment with the broad host-range bacteriophage Aquamicrobium phage P14. We incubated the phage, which was previously isolated and enriched with the Alphaproteobacteria Aquamicrobium H14, with the Betaproteobacteria Alcaligenaceae H5. During the experiment, we observed an increase in the phage's predation efficacy towards Alcaligenaceae H5. Furthermore, genome analysis and the comparison of the bacteriophage's whole genome indicated that rather than being scattered evenly along the genome, mutations occur in specific regions. In total, 67% of the mutations with a frequency higher than 30% were located in genes that encode tail proteins, which are essential for host recognition and attachment. As control, we incubated the phage with the Alphaproteobacteria Aquamicrobium H8. In both experiments, most of the mutations appeared in the gene encoding the tail fiber protein. However, mutations in the gene encoding the tail tubular protein B were only observed when the phage was incubated with Alcaligenaceae H5. This highlights the phage's tail as a key player in its adaptation to different hosts. We conclude that mutations in the phage's genome were mainly located in tail-related regions. Further investigation is needed to fully characterize the adaptation mechanisms of the Aquamicrobium phage P14.

Genome Analysis of a Novel Broad Host Range Proteobacteria Phage Isolated from a Bioreactor Treating Industrial Wastewater.

Bacteriophages are viruses that infect bacteria, and consequently they have a major impact on the development of a microbial population. In this study, the genome of a novel broad host range bacteriophage, Aquamicrobium phage P14, isolated from a wastewater treatment plant, was analyzed. The Aquamicrobium phage P14 was found to infect members of different Proteobacteria classes (Alphaproteobacteria and Betaproteobacteria). This phage contains a 40,551 bp long genome and 60% of its genes had blastx hits. Furthermore, the bacteriophage was found to share more than 50% of its genes with several podoviruses and has the same gene order as other polyvalent bacteriophages. The results obtained in this study led to the conclusion that indeed general features of the genome of the Aquamicrobium phage P14 are shared with other broad host range bacteriophages, however further analysis of the genome is needed in order to identify the specific mechanisms which enable the bacteriophage to infect both Alphaproteobacteria and Betaproteobacteria.

Multiple micro-predators controlling bacterial communities in the environment.

Predator-prey interactions are a main issue in ecological theory, including multispecies predator-prey relationships and intraguild predation. This knowledge is mainly based on the study of plants and animals, while its relevance for microorganisms is not well understood. The three key groups of micro-predators include protists, predatory bacteria and bacteriophages. They greatly differ in size, in prey specificity, in hunting strategies and in the resulting population dynamics. Yet, their potential to jointly control bacterial populations and reducing biomass in complex environments such as wastewater treatment plants is vast. Here, we present relevant ecological concepts and recent findings on micropredators, and propose that an integrative approach to predation at the microscale should be developed enabling the exploitation of this potential.

The 1.8 Å crystal structure of ACTIBIND suggests a mode of action for T2 ribonucleases as antitumorigenic agents.

ACTIBIND and its human homologue RNASET2 are T2 ribonucleases (RNases). RNases are ubiquitous and efficient enzymes that hydrolyze RNA to 3' mononucleotides and also possess antitumorigenic and antiangiogenic activities. Previously, we have shown that ACTIBIND and RNASET2 bind actin and interfere with the cytoskeletal network structure, thereby inhibiting cell motility and invasiveness in cancer and in endothelial cells. We also showed that ACTIBIND binds actin in a molar ratio of 1:2. Here, we further characterize ACTIBIND and determine its crystal structure at 1.8 Å resolution, which enables us to propose two structural elements that create binding sites to actin. We suggest that each of these binding sites is composed of one cysteine residue and one conserved amino acid region. These binding sites possibly interfere with the cytoskeleton network structure and as such may be responsible for the antitumorigenic and antiangiogenic activities of ACTIBIND and its human analogue RNASET2.

Coexistence of flexibility and stability of proteins: an equation of state.

We consider a recently suggested "equation of state" for natively folded proteins, and verify its validity for a set of about 5800 proteins. The equation is based on a fractal viewpoint of proteins, on a generalization of the Landau-Peierls instability, and on a marginal stability criterion. The latter allows for coexistence of stability and flexibility of proteins, which is required for their proper function. The equation of state relates the protein fractal dimension d(f), its spectral dimension d(s), and the number of amino acids N. Using structural data from the protein data bank (PDB) and the Gaussian network model (GNM), we compute d(f) and d(s) for the entire set and demonstrate that the equation of state is well obeyed. Addressing the fractal properties and making use of the equation of state may help to engineer biologically inspired catalysts.

The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium-loaded state.

We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold-adapted serine protease produced by Antarctic bacilli, at 1.4 A resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 A resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 A. The final models for S41 and Sph include a calcium-loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium-binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium-binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures.

Binding assay and preliminary X-ray crystallographic analysis of ACTIBIND, a protein with anticarcinogenic and antiangiogenic activities.

ACTIBIND is a T2 RNase extracellular glycoprotein produced by the mould Aspergillus niger B1 (CMI CC 324626) that possesses anticarcinogenic and antiangiogenic activities. ACTIBIND was found to be an actin-binding protein that interacts with rabbit muscle actin in a 1:2 molar ratio (ACTIBIND:actin) with a binding constant of 16.17 x 10(4) M(-1). Autoclave-treated ACTIBIND (EI-ACTIBIND) lost its RNase activity, but its actin-binding ability was conserved. ACTIBIND crystals were grown using 20% PEG 3350, 0.2 M ammonium dihydrogen phosphate solution at room temperature (293 K). One to four single crystals appeared in each droplet within a few days and grew to approximate dimensions of 0.5 x 0.5 x 0.5 mm after about two weeks. Diffraction studies of these crystals at low temperature (100 K) indicated that they belong to the P3(1)21 space group, with unit-cell parameters a = 78, b = 78, c = 104 A.