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.

FIGURE 1.

The strategy used for the production of d-Lac-ended peptidoglycan precursors in L. lactis. d-Ala-d-Lac was produced in L. lactis by expressing the ddl_Lp ligase from L. plantarum. In a second step, the endogenous ddl_Lc ligase of L. lactis was inactivated to reduce the level of precursors ended by d-Ala-d-Ala. The complete substitution of d-Ala-ended precursors by d-Lac-ended precursors was achieved by expressing the Aad dipeptidase that eliminates d-Ala-d-Ala dipeptides produced by ddl_Lp (dashed lines). MurF, uridinediphospho-n-acetylmuramyl-tripeptide:d-Ala-d-Lac ligase; tri, l-Ala-d-Glu-m-DAP; hexagon, n-acetylmuramic acid.

FIGURE 2.

Growth dependence of the MD001 and MD003 mutants toward nisin and d-lactate. The L. lactis MD001 (A) and MD003 (B) mutants were cultured in M17-glucose in the presence and absence of d-Lac (15 mm) and nisin (0.5 ng/ml), shown as closed and open triangles, respectively, and in the presence of nisin (0.5 ng/ml) without d-Lac (open circles). Growth of the cultures was followed by measuring the A_{600 nm}.

FIGURE 3.

Phenotypic adaptation to d-Lac-ended peptidoglycan precursors in L. lactis. A, shown are representative vancomycin antibiograms obtained for the control strain MD006, the primary mutant MD003, and its vancomycin-resistant derivative MD003-128 by the E-test method (see “Experimental Procedures”). Arrows indicate the presence of highly resistant colonies in the E-test inhibitory zone obtained for MD003. B, evolution of vancomycin resistance within cell populations of the MD006 (top) and MD003 (bottom) primary mutants is shown. Dilutions of serial cultures of both mutant strains were plated on increasing concentrations of vancomycin. Graphs express the number of colony forming units (CFU) per ml and A_{600 nm} units of the cultures obtained at each vancomycin concentration after 10 (squares), 20 (circles), 30 (triangles), and 40 (diamonds) generations. C, shown is growth rate comparison between the MD003 primary mutant and its vancomycin-resistant derivative MD003-128. Three independent cultures of the control strain MD006, the vancomycin-resistant clone MD003-128, and the MD003 primary isolate were performed in M17-glucose supplemented with erythromycin (5 μg/ml), chloramphenicol (5 μg/ml), d-Lac (20 mm), and nisin (0.5 ng/ml). Growth of the cultures was followed by measuring the A_{600 nm} as a function of time. For MD006 (squares) and MD003-128 (circles), mean values obtained for the three cultures are plotted. For MD003 (triangles), the growth curves obtained for the three independent cultures are shown.

FIGURE 4.

Effect of changing the precursor on peptidoglycan structure. Peptidoglycan extracted from the reference strain PH8076 (pNZ8048) (top) and from the d-Lac-ended precursor-producing MD003 mutant (bottom) were digested with the mutanolysin muramidase and analyzed by HPLC as described under “Experimental Procedures.” The chemical structures assigned to the identified picks of muropeptides (numbered) are given in supplemental Table S3 and Table 2.

FIGURE 5.

Van-FL staining and morphology of L. lactis mutant strains expressing different proportions of d-Lac-ended versusd-Ala-ended peptidoglycan precursors. For each strain micrographs of representative cells visualized by Van-FL staining (upper panels) and 4′,6-diamidino-2-phenylindole staining (lower panels) are presented. Scale bars: 1 μm. A, wild type NZ3900 cells transformed with the empty expression vector pNZ8048 are shown. Cells are numbered according to cell cycle progression. Arrowheads show brighter fluorescence spots that appear at the division site and then separate at a later stage of cell division to occupy a polar position in the newborn cells. A schematic representation of the different stages of the cell cycle is shown on the left, with the Van-FL-stained zones and spots colored gray. B, cells of the MD001 mutant producing a mixture of d-Ala-ended and d-Lac-ended peptidoglycan precursors are shown. Arrows indicate morphological defects associated with dark areas not stained by the Van-FL probe. C, cells of the MD003 mutant exclusively producing d-Lac-ended peptidoglycan precursors are shown. Contrast of the fluorescence images was increased to visualize the position of the cell in the field. D, shown is a representative cell chain formed by the highly vancomycin resistant MD003 derivative MD003-128.

FIGURE 6.

Effect of methicillin on cell morphology. Exponentially growing cells of the control strain MD006 (A), the MD003 primary mutant (B), the highly vancomycin-resistant MD003-derived clone MD003-128, and the carboxypeptidase dacA (BLD006; D) and dacB (VES2065; E) mutants were incubated for 4h30 with the indicated concentrations of methicillin before be examined by light microscopy. Scale bar = 3μm.