AcmD, a homolog of the major autolysin AcmA of Lactococcus lactis, binds to the cell wall and contributes to cell separation and autolysis

Abstract

Lactococcus lactis expresses the homologous glucosaminidases AcmB, AcmC, AcmA and AcmD. The latter two have three C-terminal LysM repeats for peptidoglycan binding. AcmD has much shorter intervening sequences separating the LysM repeats and a lower iso-electric point (4.3) than AcmA (10.3). Under standard laboratory conditions AcmD was mainly secreted into the culture supernatant. An L. lactis acmAacmD double mutant formed longer chains than the acmA single mutant, indicating that AcmD contributes to cell separation. This phenotype could be complemented by plasmid-encoded expression of AcmD in the double mutant. No clear difference in cellular lysis and protein secretion was observed between both mutants. Nevertheless, overexpression of AcmD resulted in increased autolysis when AcmA was present (as in the wild type strain) or when AcmA was added to the culture medium of an AcmA-minus strain. Possibly, AcmD is mainly active within the cell wall, at places where proper conditions are present for its binding and catalytic activity. Various fusion proteins carrying either the three LysM repeats of AcmA or AcmD were used to study and compare their cell wall binding characteristics. Whereas binding of the LysM domain of AcmA took place at pHs ranging from 4 to 8, LysM domain of AcmD seems to bind strongest at pH 4.

Figure 1. Modular architecture of AcmA, AcmD and the fusion proteins used in this study.

The description of the protein is depicted on the left and their respective molecular masses (MM) (kDa) and pIs are given on the right. Dark and light grey boxes indicate the LysM repeats and their intervening amino acid sequences, respectively. *, site of in-frame myc-tag insertion in the active site of AcmD; MSA-2, Plasmodium falciparum Merozoite Surface Antigen-2; GFP, Green Fluorescence Protein; His_10, tag of 10 Histidine residues; SS, Signal Sequence; ppPrtP, pre-pro of prtP.

Figure 2. AcmD contributes to cell autolysis.

Determination of cell lysis upon induced expression of AcmD. Cultures of L. lactis NZ9000 (pNGacmD) or L. lactis NZ9000acmA∆1 (pNGacmD) were grown until an OD₆₀₀ of 0.6 was reached. Both cultures were split in two. Expression of AcmD was induced in one half of each culture by the addition of nisin (10 ng/ml), the other halves were left un-induced. The cells of the four cultures were collected after 2 h and mixed with the cell-free culture supernatant of L. lactis MG1363 (dark grey bars) or L. lactis MG1363acmA∆1 (grey bars). After 1 h incubation at 30° C, supernatants were analyzed for PepX activity (in arbitrary units (AU)). The slope of the substrate hydrolysis/color development was determined and is indicated as PepX activity (AU)/min. The average was calculated from two independent experiments.

Figure 3. AcmD of L. lactis is involved in cell separation.

(a) Turbidity and cell sedimentation of L. lactis IL1403 (1), IL1403acmA::ISS1 (2) and IL1403acmA::ISS1acmD::myc (3) after overnight growth at 30° C in GM17 broth. (b) Light microscopic views of L. lactis IL1403, IL1403acmA::ISS1 and IL1403acmA::ISS1acmD::myc after overnight growth at 30° C as standing cultures in GM17 medium. Magnification: 1250x in all three frames; representative views. (c) Phase contrast microscopic views of uninduced (left) and nisin (10 ng/ml)-induced (right) IL1403acmA::ISS1acmD::myc harboring plasmids pNGAcmD (AcmD) and pNZ9530 (nisRK). (d) Quantification of chain length in L. lactis IL1403, IL1403acmA::ISS1, IL1403acmA::ISS1acmD::myc, and nisin (10 ng/ml)-induced IL1403acmA::ISS1acmD::myc (pNGacmD) (the complemented double mutant). Number of diplococci per chain was counted and the mean of 30 chains were depicted. Standard deviation (***) indicates the significance as analyzed by Bonferroni’s multiple comparison test (p<0.05) using one-way ANOVA.

Figure 4. Binding of MSA2-LysM_AcmA and MSA2-LysM_AcmD to L. lactis cells.

a. Anti-MSA2 antibody-treated Western blot showing binding of MSA2-LysM_AcmA, MSA2-LysM_AcmD and MSA2 to non-treated (lanes 1, 2 and 3) and TCA-treated (lanes 4, 5 and 6) L. lactis cells, respectively. b. Western blot treated with anti-MSA2 antibody. Lanes 1 and 3 indicate MSA2-LysM_AcmD bound to TCA-treated L. lactis cells at pH 6.2 and 3.2, respectively. The unbound protein at pH 6.2 and 3.2 is shown in lanes 2 and 4, respectively. Lane 5, positive control: MSA2-LysM_AcmA bound to L. lactis at pH 6.2. Arrow points out Pro-MSA2-LysM_AcmD and * indicates mature MSA2-LysM_AcmD. The percentage of both MSA2-LysM variants bound to L. lactis cells at pH 6.2 and 3.2 were semi-quantified (as the number of pixels present per mm² of both bands in lanes 1 or 3) divided by the total signal of the cell and supernatant fractions (number of pixels per mm² of all bands in lanes 1+2 or 3+4, respectively) is shown at the bottom of lanes 1 and 3. c. Transmission electron microscopic images of L. lactis incubated with MSA2 or MSA2 fusion proteins and subsequently incubated with rabbit anti-MSA2 antibodies and finally decorated with goat anti-rabbit IgG gold marker. Picture 1: non-treated L. lactis cells incubated with MSA2-LysM_AcmA. Pictures 2, 3 and 4 show TCA-treated L. lactis cells incubated with MSA2-LysM_AcmA, MSA2-LysM_AcmD and MSA2 proteins, respectively. Arrows indicate immunogold particles detected on the cells.

Figure 5. Binding of LysM_AcmD-GFP-His₁₀ and LysM_AcmA-GFP-His₁₀ to L. lactis NZ9000 cells.

Phase contrast and fluorescence microscopy of L. lactis NZ9000 cells incubated with (a) LysM_AcmD-GFP-His₁₀ at pH 4.0 and (b and c) LysM_AcmA-GFP-His₁₀ at pH 4.0 and 8.0, respectively. Original magnification: 1250-fold in all frames.