Functional and Morphological Adaptation to Peptidoglycan Precursor Alteration in Lactococcus lactis

Abstract

Cell wall peptidoglycan assembly is a tightly regulated process requiring the combined action of multienzyme complexes. In this study we provide direct evidence showing that substrate transformations occurring at the different stages of this process play a crucial role in the spatial and temporal coordination of the cell wall synthesis machinery. Peptidoglycan substrate alteration was investigated in the Gram-positive bacterium Lactococcus lactis by substituting the peptidoglycan precursor biosynthesis genes of this bacterium for those of the vancomycin-resistant bacterium Lactobacillus plantarum. A set of L. lactis mutant strains in which the normal D-Ala-ended precursors were partially or totally replaced by D-Lac-ended precursors was generated. Incorporation of the altered precursor into the cell wall induced morphological changes arising from a defect in cell elongation and cell separation. Structural analysis of the muropeptides confirmed that the activity of multiple enzymes involved in peptidoglycan synthesis was altered. Optimization of this altered pathway was necessary to increase the level of vancomycin resistance conferred by the utilization of D-Lac-ended peptidoglycan precursors in the mutant strains. The implications of these findings on the control of bacterial cell morphogenesis and the mechanisms of vancomycin resistance are discussed.Peptidoglycan (or murein) is a major stress-bearing component of the bacterial cell wall. This polymer is made of glycan chains interconnected by covalent cross-links between short peptides. It acts as an exoskeleton and plays a key role in determining and maintaining cell shape throughout the cell cycle.

PBP2b plays a key role in both peripheral growth and septum positioning in Lactococcus lactis

“…pneumoniae (PBP3 - ) resulted in similar defects as observed here for the L . lactis pbp2b mutant [ 12 , 39 – 41 ]. In those mutants, peripheral growth is inhibited [ 12 , 39 – 41 ], PG outgrowths (piecrusts) could not be visualized [ 41 ], and septa are misplaced [ 12 , 41 ].…”

“…lactis [ 5 , 9 , 12 , 13 ]. Consistent with its central role in cell division, inhibition of PBP2x transpeptidase activity led to cell filamentation in both species [ 5 , 9 , 12 , 13 ], but with the production of longer filaments in L . lactis [ 9 , 12 ].…”

Influence of Polyelectrolyte Film Stiffness on Bacterial Growth

“…The pH of the medium was fixed at 6.4 and 7 (by adding 0.1 M NaOH or 0.1 M HCl) for L. Lactis and E. coli , respectively to optimize the bacterial growth conditions. 44,45 pH measurements were regularly performed in the medium during the tests to check the stability of the growth conditions. Moreover, some assays were performed while changing the CDM medium once during the experiment to assess whether this operation affects or not the bacterial multiplication onto the sample surface.…”

Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis

“…The gene dacB of MG1363 encodes a protein with L,D-carboxypeptidase activity that is involved in PG maturation (Courtin et al 2006). Another D-Ala-D-Ala carboxypeptidase (DacA) of MG1363 converts a pentapeptide side chain in PG to a tetrapeptide side chain (Deghorain et al 2010). Both dacA and dacB are present in IL1403.…”