Etude du rôle fonctionnel des isomères du lactate chez Lactobacillus plantarum par une approche génétique
(2004)
Files
Goffin.pdf
Restricted Access - Adobe PDF
- 63.63 MB
Details
- Authors
- Supervisors
- Abstract
- Lactobacillus plantarum belongs to the group of lactic acid bacteria, whose fermentative metabolism is focused on lactate production. It produces both D- and L-lactate in a ratio close to the equivalence through the reduction of glycolytic pyruvate, catalyzed by two stereospecific enzymes, the NAD-dependent D- and L-lactate dehydrogenases (LdhD and LdhL). In addition, previous work reported the presence of an additional pathway for D-lactate production in this species. This pathway was suggested to be an L-lactate inducible lactate racemase, as already observed in other lactic acid bacteria. Yet, no information was available regarding the genetic determinants of this activity in any species. The present work was thus aimed at studying the pathway of lactate racemization in L. plantarum by answering the following questions : What is the mechanism of lactate racemization in L. plantarum ? What is the physiological role of such an activity in a species that already possesses all the enzymes required for both D- and L-lactate production and utilization ? What is the role of D-lactate in the physiology of L. plantarum ? We first showed that the lactate racemization activity of L. plantarum was independent of any lactate dehydrogenase activity (NAD-dependent or independent). This required the cloning and knock-out of the loxD and loxL genes, encoding the NAD-independent D- and L-lactate dehydrogenases, respectively. The loxD and loxL knock-out mutants, together with the previously described ldhD and ldhL mutant strains, were then used to investigate the lactate utilization pathway in the stationary phase of aerobic growth of L. plantarum. The NAD-dependent Ldh enzymes were found to be responsible for this lactate consumption, while the role of NAD-independent Lox enzymes remains unknown. Using a global transcriptomic approach, we identified a six-genes operon, named lar, that was highly induced by L-lactate. The involvement of this operon in lactate racemase activity was shown through the construction of knock-out and over-expressing mutants. It was also demonstrated that lactate racemase activity was responsible for D-lactate production in the absence of LdhD activity, thereby supporting its role as a rescue pathway for D-lactate production. The physiological role of D-lactate in L. plantarum was thus investigated by constructing a strain producing exclusively L-lactate. This was achieved through the combined inactivation of both D-lactate production pathways, the LdhD and lactate racemase activities. The effects of disrupting D-lactate production in L. plantarum were then investigated. D-lactate is known to be a key component of the peptidoglycan in this species, where it is incorporated at the last position of the pentadepsipeptide, leading to natural vancomycin resistance. In our work, particular attention was thus paid to peptidoglycan synthesis and vancomycin resistance in this homo-L-lactic strain. In our work, we were able to show that D-lactate is absolutely required for the growth of L. plantarum, and that this requirement is due to the apparent inability of the peptidoglycan synthesis machinery to efficiently synthesize and incorporate peptidoglycan precursors terminating in D-2-aminoacids.
- Affiliations
Citations
Goffin, P. (2004). Etude du rôle fonctionnel des isomères du lactate chez Lactobacillus plantarum par une approche génétique. https://hdl.handle.net/2078.5/97971