Martijn Bekker

Comparative Systems Biology: Lactic Acid Bacteria

"Systems Understanding of Microbial Oxygen responses" (SUMO) investigates how Escherichia coli senses oxygen, or the associated changes in oxidation/reduction balance, via the Fnr and ArcA proteins, how these systems interact with other regulatory systems, and how the redox response of an E. coli population is generated from the responses of single cells. There are five sub-projects to determine system properties and behaviour and three sub-projects to employ different and complementary modelling ...

Here SYSMO-LAB put all there pre-liminary data files or models ordered per person and project

Challenge: Comparative analyses, as demonstrated by comparative genomics and bioinformatics, are extremely powerful for (i) transfer of information from (experimentally) well-studied organisms to the other organisms, and (ii) when coupled to functional and phenotypic information, insight in the relative importance of components to the observed differences and simalities. The central principle of this proposal is that important aspects of the functional differences between organisms derive not ...

Here you will find all pre-liminary data

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 ...

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

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

Enzymes involved in butanediol formation from pyruvate in L. Lactis and E. faecalis were characterized with respect to their Km's for their substrates and their Vmaxes

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.

In this experiment we glucose-pulsed an E. faecalis 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 catqabolism of E. faecalis

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

L. lactis cultures were grown at different dilution rates in glucose-limited chemostat conditions and were analyzed with respect to physiological parameters. Amino acid consumption, glucose consumption and production of fermentation products were measured in steady-state conditions,

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

S. pyogenes was grown in C-limited cultures at pH 6.5 and 7.5 and at a growth rate of 0.05 The glnA mutant strain shows decreased growth in low glutamine and excess glutamate conditions and no growth at all in low glutamine and low glutamate conditions. Preliminary results of glucose-limited chemostat cultures indicate a reversion of the pH dependency of the shift from homolactic to more mixed acid fermentation: wild type - lactate/formate ratio at pH 6.5 = 11.8, at pH 7.5 = 2.8 glnA mutant - ...

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

qATP values were calculated based on fermentation product formation and expected used biochemical pathways. These were averaged and used for calculation of the ATP required for maintenance and for growth. These data were subsequently used to calculate the qATP at the maximal growth rate by extrapolation

E. faecalis was gucose-pulsed after resuspension in 100 mM MES buffer at pH 6.5 Intra- and extracellular metabolites concentrations were 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.

Method extraction of intracellular metabolites in Lactococcus lactis

Method extraction of intracellular metabolites in Lactococcus lactis

Method for transformation of plasmids into Lactococcus lactis

Method for synthesis of LAB-medium sued for the SYSMO-LAB project

Method for analysis of various organic acids in the medium

Protocol for applying a glucose perturbation in Streptococcus pyogenes.

Perturbation of starved cells with glucose. Concentrations of intra- and extracellular metabolites are followed in time.

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