Investigation of glycolysis and pyruvate branching of three lactic acid bacteria

SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".

The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.

Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...

Comparative Systems Biology: Lactic Acid Bacteria

The reconstruction of the metabolic networks is done by sequence comparison with already annotated genomes of L. lactis, L. plantarum, B. subtilis and E. coli

The two lactic acid bacteria L. lactis and S. pyogenes were studied with respect to the concentration of intracellular metabolites involved in glycolysis in time upon a glucose pulse. Models that describe this behavior are also constructed

Lactic acid bacteria generally use homolactic fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three lactic acid bacteria Lactococcus lactis, Enterococcus faecalis and Streptococcus pyogenes. Surprisingly deletion of the ldh genes hardly affected the growth rate in chemically defined medium, however growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various ...

The Lactate dehydrogenases (LDH) are key metabolic enzymes in lactic acid bacteria (LAB). The LDH ( E.C. 1.1.1.27) catalyzes the reaction of pyruvate and NADH into lactate and NAD+.We have carried out an experimental and computational study of the effects of fructose-1,6-bisphosphate (FBP), phosphate (Pi) and ionic strength (NaCl concentration) on 3 LDHs from 3 LABs studied at pH 6 and pH 7.

S. pyogenes was grown in rich medium, strongly concentrated and glucose-pulsed in a MES buffer. Intracellular metabolite concentration is followed in time.

S. pyogenes M49 (591), E. faecalis V583, and L. lacis NZ9000 and their isogenic ldh deletion mutants were grown glucose free CDM-LAB medium in BIOLOG phenotype microarray plates PM01 and PM02. With this assay the abilitiy of the strains to grow on 190 different carbon sources was determined in 96 well format.

L. lactis was grown in LAB medium or rich THY medium, strongly concentrated and glucose-pulsed in a MES buffer. Intracellular metabolite concentration is followed in time.

Measurements on Km, Vmax and allosteric activation or inhibition of the heterologously expressed (E. coli) and purifiied main L-lactate dehydrogenase

Measurements on Km, Vmax and allosteric activation or inhibition of the main L-lactate dehydrogenase

Global sensitivity analysis of a kinetic model to determine the sensitivities for each parameter, over a wide parameter range. We used the elementary effects method.

Lumped kinetic model of L. lactis glycolysis, formulated with ordinary differential equations. Simulations are in line with experimental data

L. lactis, E. faecalis and S. pyogenes are referred to as LAB because of the fact that in the presence of glucose lactate is produced as the main fermentation product . This metabolic pathway is relatively inefficient, since only 2 ATP are generated from one glucose molecule. All three LAB possess the genetic make up for mixed acid fermentation, a more effective way of fermentation generating 3 ATP per molecule of glucose. All three genomes reveal (at least) two genes encoding a lactate dehydrogenase ...

L. lactis, S. pyogenes and E. faecalis were grown in C-limited chemostat cultures at various pH's and dilution rates. General flux distribution, yields and other physiological factors were studied.

Metabolic network of S. pyogenes including primary metabolism, polysaccharide metabolism, purine and pyrimidine biosoynthesis, teichoic acid biosynthesis, fatty acid and phospholipid bioynthesis, amino acid metabolism, vitamins and cofactors

Metabolic network of Enterococcus faecalis including primary metabolism, polysaccharide metabolism, purine and pyrimidine biosoynthesis, teichoic acid biosynthesis, fatty acid and phospholipid bioynthesis, amino acid metabolism, vitamins and cofactors

Despite high similarity in sequence and catalytic properties, the L-lactate dehydrogenases (LDH) in lactic acid bacteria (LAB) display differences in their regulation which may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose-1,6-bisphosphate (FBP), phosphate (Pi) and ionic strength (NaCl concentration) on 6 LDHs from 4 LABs studied at pH 6 and pH 7. We find: (1) The extent of activation by FBP (Kact) ...

Output of the 3D-structures modeled by comparative modeling tool for LDH enzymes from four LABs (in the PDB format, tarred). Four LABs include Enterococcus faecalis, Lactococcus lactis, Streptococcus pyogenes and Lactobacillus plantarum. Output of the SEEK Model https://seek.sysmo-db.org/models/118.

The modeling was performed against a x-ray structure of LDH from B. stearothermophilis (template, PDH ID: 1LDN).

Output files of phosphate probe binding on the surface of LDH from lactococcus lactis type 1. File with extension XPLOR can be visualized with a program VMD to identify the most favorable position for the phosphate binding. This relates to the Model "Part 4".

The Table represents the simulation results of how the presence of phosphate ions (Pi) in the solution might affect the activity of four LDH enzymes. This includes the algorithmic analysis of the binding energies values computed by the GRID program (see Part 4, model) for each enzyme in presence and absence of FBP molecule at pH 6 and 7. The analysis was performed by using the algorithm proposed in Part 5, model.

L. lactis was grown in rich THY medium, strongly concentrated and glucose-pulsed in a MES buffer. Intracellular metabolite concentration is followed in time.

. L. lactis (NZ9000), E. faecalis (V538) and S. pyogenes (M49) wild type strain and their ldh- mutants were grown in batch cultures at 37°C in anaerobic 96 wells plates in either TH-broth supplemented with 0.5% (w/v) yeast (THY) or a chemically defined medium for LAB (pH 7.4) (CDM-LAB (10)). Both media were buffered with either 100 mM MES buffer or 100 mM MOPS buffer for growth at pH 6.5 and 7.5 respectively.

S. pyogenes was grown in C-limited cultures at pH 6.7 and 7.5 and at a growth rate of 0.05 and 0.15

E. faecalis was grown in C-limited cultures at pH 6.7 and 7.5 and at a growth rate of 0.05 and 0.15

L. lactis was grown in C-limited cultures at pH 6.7 and 7.5 and at a growth rate of 0.05, 0.15 and 0.40

In this experiment we glucose-pulsed an L. lactiss cultures re-suspended in 100 mM MES buffer at pH 6.5. Samples were taken in time to study intra- and extracellular metabolites. These data are used to construct a kinetic model of the catabolism of E. L. lactis

Glucose pulsed S pyogenes with 5 mM glucose in 100 mM MES buffer at pH 6.5 and followed intracellular metabolites in time

Glucose pulsed L. lactis with 20 mM glucose in 100 mM MES buffer at pH 6.5 and followed intracellular metabolites in time

L. lactis was grown in LAB medium, strongly concentrated and glucose-pulsed in a MES buffer. Intracellular metabolite concentration is followed in time.

For each modeled 3D structure of LHD (see Part 1: 3D structure modeling for LDH enzymes) was computed electrostatic potential by using the UHBD program. The files are in GRD format (binary) and can be visualized with the graphical programs as CHIMERA or VMD.

The output includes the similarity matrix of LDH enzymes based on comparison of the electrostatic potentials at allosteric and catalytic binding sites, separately. The similarity indices were generated by the PIPSA program (http://projects.villa-bosch.de/mcmsoft/pipsa/3.0/).

Heterologous Expression of LDHs from different lactic acid bacteria in Escherichia coli DH5α. Assessment of kinetic parameters of LDH to include in a catabolic model .

Heterologous Expression of LDHs from different lactic acid bacteria in Escherichia coli DH5α.

Assessment of kinetic parameters of LDH to include in a catabolic model.

Preliminary metabolic network of S. pyogenes including primary metabolism, polysaccharide metabolism, purine and pyrimidine biosoynthesis, teichoic acid biosynthesis, fatty acid and phospholipid bioynthesis, amino acid metabolism, vitamins and cofactors. The model still needs to be validated.

A reconstruction of the cellular metabolism of the opportunistic human pathogen Enterococcus faecalis V583 represented as stoichiometric model and analysed using constraint-based modelling approaches

3D structure prediction of LDH enzymes from four LAB by comparative modeling against x-ray structure of LDH from B. stearothermophilis (template, PDB ID: 1LDN). The computation was performed with a protocol that uses "automodel.very_fast" settings of Modeller program (http://salilab.org/modeller/).

Comparison of electrostatic potentials within the allosteric binding sites of LDH enzymes to estimate the binding affinity of the FBP molecule is performed with the PIPSA program. The program uses the structure of enzymes in the PDB format and computed electrostatic potentials in the GRD format.

Computation is performed for the modeled 3D structures of LDH enzymes (in PDB format) with the UHBD program, for pH 6 and pH 7.

Binding energies of phosphate ions to the allosteric and catalytic sites were estimated with a program GRID (http://www.moldiscovery.com/soft_grid.php). The calculations were performed for the modeled LDH structures from four LABs, at pH 6 and 7, in presence and absence of the FBP molecule. The phosphate ion was presented as a probe.

In order to estimate whether Pi has an activatory or an inhibitory effect on the enzymes, the computed probe binding energies (from GRID results, Part 4) were compared with those for the LDH from L. plantarum whose activity is known to be unaffected by Pi.

The binding energies of the Pi probe in the allosteric binding site (AS) and the COO probe in the catalytic binding site (CS) of LDH from L. plantarum were defined as E¬AS,threshold and ECS,threshold, respectively. For the other LDH enzymes, ...

Method extraction of intracellular metabolites in Lactococcus lactis

This HPLC method uses a isocratic method and a RI detector to identify and quantify almost all excreted catabolic metabolites.

A protocol for acidic quenching of lactic acid bacteria used for analyses of intracellular metabolites.

This protocol for applying glucose perturbations works for Lactococcus lactis and Enterococcus faecalis