Molecular insights into the metabolism and physiology of the lactic acid bacterium "Lactobacillus delbrueckii" subsp. "lactis"

In the course of this doctorate work, we gained insights into the energy metabolism of Lactobacillus delbrueckii subsp. lactis by purifying and characterising 3-phosphoglycerate kinase (PGK) and pyruvate kinase (PYK). These two enzymes are the two enzymes of the Embden-Meyerhof pathway able to regenerate ATP as part of glycolysis. Since L. lactis relies on substrate phosphorylation for energy production, the role of PGK and PYK is particularly important in this bacterium. 3-phosphoglycerate kinase is a 45 kDa–monomer and a Michaelis-Menten type enzyme with Km of 2.6 mM for 3-phosphoglycerate and 0.7 mM for ATP. As the enzymatic assays used for the characterisation of this enzyme go in the reverse direction of glycolysis, no further study was performed. Pyruvate kinase, on the other hand, was characterised in more depth. PYK is an homotetramer with a subunit MW= 68 kDa, and an allosteric enzyme. It is activated by the glycolysis precursors, fructose 1,6-diphosphate, fructose 6-phosphate and glucose 6-phosphate, and inhibited by high concentrations of PEP, inorganic phosphate and ATP. An ATP binding motif present on a 112 residue–C-terminal extension of the enzyme was purported to be implicated in the regulation of PYK by ATP, thus reinforcing the role of pyruvate kinase as a key modulator of L. lactis glyolysis and energy metabolism. In a second stage of this project, we concentrated on the study of restriction-modification (R-M) systems in L. lactis. The first indications of the presence of R-M systems came from the analysis of two plasmids, pN42 and pJBL2, isolated from L. lactis strains NCC88 and JBL2, respectively, and were two-fold: (i) evidence of modification catalysed by an N6methyladenine methyltransferase that may be part of a R-M system, (ii) presence of an hsdS gene on each of the two L. lactis plasmids, suggesting the presence of a complete type I hsd cluster on the chromosome. Purification attempts yielded a semi-purified enzyme fraction displaying activity characteristic of a type I restriction enzyme. A combination of PCR and inverted PCR techniques was implemented to isolate type I hsd clusters from two L. lactis strains NCC88 and NCC82. These hsd clusters encode genes that are highly conserved (>97% identity) except for the hsdS genes that have different specificities. The hsd clusters could be divided into two parts. The genes hsdR, hsdM, and hsdS organised in two transcriptional units, the single gene hsdR regulon and the hsdMS operon constitute the first part of L. lactis hsd clusters and what we referred to as the ‘hsd cluster proper’ since they are the units constituting hsd clusters isolated and characterised in E. coli. L. lactis hsd clusters comprise a second part located downstream of the hsd cluster proper encoding and integrase (int) gene as well as a second N-terminally truncated hsdS gene. Comparison of the hsdS genes belonging to the
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