Characterization of SCO4439, a D-alanyl-D-alanine carboxypeptidase involved in spore cell wall maturation, resistance, and germination in Streptomyces coelicolor

Abstract

This work contributes to the understanding of cell wall modifications during sporulation and germination in Streptomyces by assessing the biological function and biochemical properties of SCO4439, a D-alanyl-D-alanine carboxypeptidase (DD-CPase) constitutively expressed during development. SCO4439 harbors a DD-CPase domain and a putative transcriptional regulator domain, separated by a putative transmembrane region. The recombinant protein shows that DD-CPase activity is inhibited by penicillin G. The spores of the SCO4439::Tn5062 mutant are affected in their resistance to heat and acid and showed a dramatic increase in swelling during germination. The mycelium of the SCO4439::Tn5062 mutant is more sensitive to glycopeptide antibiotics (vancomycin and teicoplanin). The DD-CPase domain and the hydrophobic transmembrane region are highly conserved in Streptomyces, and both are essential for complementing the wild type phenotypes in the mutant. A model for the biological mechanism behind the observed phenotypes is proposed, in which SCO4439 DD-CPase releases D-Ala from peptidoglycan (PG) precursors, thereby reducing the substrate pool for PG crosslinking (transpeptidation). PG crosslinking regulates spore physical resistance and germination, and modulates mycelium resistance to glycopeptides. This study is the first demonstration of the role of a DD-CPase in the maturation of the spore cell wall.

Figure 1. Analysis of the germination stages in S. coelicolor wild type and in S. coelicolor SCO4439::Tn5062 mutant.

(a–l) Confocal laser-scanning fluorescence microscopy analysis (SYTO9/PI staining) of the S. coelicolor wild type (left panel) and SCO4439::Tn5062 mutant (right panel). The same samples were observed using the fluorescence (left pictures) or interference contrast modes (right pictures). Bars indicate 8 μm (m,n) Time-lapse confocal microscopy (interference contrast mode) of the germination of spores from the wild type and of the SCO4439::Tn5062 mutant, respectively. Spore diameters are indicated. (o,p) Confocal laser-scanning fluorescence microscopy analysis (SYTO9/PI staining) and interference contrast images of spore swelling and spore deswelling stages in the wild type and SCO4439::Tn5062 mutant, respectively. (q) Time-lapse confocal microscopy of the germination of spores from the SCO4439::Tn5062 mutant harboring the SCO4439* mutated gene (SCO4439::Tn5062[pBRB3*] strain). Time-lapse experiments were limited to 12 hours (see Methods for details). Arrows indicate spores. Asterisks indicate swelled spores. (r) Quantification of the spore diameters (average ± SD) of the wild type, SCO4439::Tn5062 mutant, and SCO4439::Tn5062[pBRB3*] strain at 5, 8 and 15 hours.

Figure 2. Complementation of the wild-type phenotype in the SCO4439::Tn5062 mutant, and SCO4439 gene expression during development.

(a) Scheme of the SCO4437-SCO4442 region in the wild type and the SCO4439::Tn5062 mutant. The dashed line indicates the chromosome deletion generated by the transposon insertion. (b) Scheme illustrating the fragments introduced into the mutant strain using plasmids pBRB1, pBRB2 and pBRB3. Fragments that complemented the phenotype in the mutant strain are highlighted in red. (c) Confocal laser fluorescence microscopy analysis of the S. coelicolor wild-type strain harboring pMS82 (control), and the S. coelicolor SCO4439::Tn5062 mutant strain harbouring pBRB3 (complementation of the phenotype is also observed with pBRB2 but not with pBRB1). Samples were observed using the fluorescence (left pictures) or interference contrast modes (right pictures). Arrows indicate spores. (d) Quantification of spore diameters (average ± SD) in the wild type harboring pMS82 and in the SCO4439::Tn5062 mutant harboring pBRB3. (e) SCO4439 gene abundance at 12, 24, 48 hours (aerial mycelium) and 72 hours (spores). Data represent the fold change with respect to the 12 hour time point (qRT-PCR and microarray values). Microarray data are from Yagüe et al..

Figure 3. Spore resistance to physicochemical stresses and mycelium resistance to glycopeptides (vancomycin and teicoplanin).

(a) Spore resistance to freezing. (b) lysozyme. (c) sonication. (d) acid. (e) heating. (f) Minimum inhibitory concentrations of vancomycin and teicoplanin towards S. coelicolor. The (1) effect on germination of the treated spores with respect to the untreated spores, and the (2) MIC values were estimated for the S. coelicolor wild-type strain (wt), SCO4439::Tn5062 mutant (SCO4439 mutant), SCO4439::Tn5062 complemented with pBRB3 (complemented mutant) and the control wild type strain harboring pMS82 (wt pMS82). Note that lysozyme treatment increased germination in all strains. Asterisks indicate a significant increase in resistance to acid and heating in the mutant strain. Percentages of germination are the average ± SD of three replicates; MIC values were estimated using three biological replicates; SD = 0.

Figure 4. Structure of SCO4439 and orthologous proteins in the Streptomyces genus.

(a) Scheme indicating the structure of the SCO4439 protein. Conserved database domain (CDD) references and their average similarities in the Streptomyces genus are indicated. SCO4439 fragments introduced in plasmids pBRB3, pBRB4, pBRB5 and pBRB6 are shown schematically. Fragments that complemented the phenotype in the mutant strain are highlighted in red. (b) Sequence alignment of the DD-CPase domain of SCO4439 (S. coelicolor) and their orthologs in other model streptomycetes. Conserved “SxxK”, “SxN” and “KTG” motifs that characterize the “SxxK” superfamily of DD-peptidases are indicated. An asterisk indicates the Leu₆₈₄ whose replacement by Pro partially blocks complementation of the wild type phenotype in the SCO4439::Tn5062 mutant.

Figure 5. Purification of recombinant His₆-SCO4439/ His₆-SCO4439*, SCO4439 activity and cellular localization.

(a) Coomassie-stained SDS-PAGE gel of overproduced His₆-SCO4439 and His₆-SCO4439* (substitution of Leu₆₈₄ for Pro₆₈₄). M, molecular weight markers. Lane 1, E. coli JM109 producing His₆-SCO4439 (45 μg). Lane 2, E. coli JM109 producing His₆-SCO4439* (45 μg). Lane 3, purified His₆-SCO4439 (4 μg). Lane 4 purified His₆-SCO4439* (4 μg). (b) DD-CPase and DD-dipeptidase activities of His₆-SCO4439 and His₆-SCO4439*. Enzyme activity values (units per mg of pure recombinant protein) are the average ± SD of three replicates. (c) His₆-SCO4439 penicillin inhibition curve. (d,e) DD-CPase and DD-dipeptidase activity (units per mg of total protein) detected in insoluble fractions (membrane and cell wall debris) of S. coelicolor wild type and of SCO4439::Tn5062 mutant strains. (f) PG crosslinking in the spores of the SCO4439::Tn5062 mutant (SCO4439 mutant), S. coelicolor wild type (wt), SCO4439::Tn5062 complemented with pBRB3 (complemented mutant), the control wild type strain harboring pMS82 (wt pMS82), and the SCO4439::Tn5062 mutant complemented with pBRB3* (SCO4439 mutant [pBRB3*]). Values are the means ± SD of two biological replicates, and four technical replicates.

Figure 6. Nascent-PG synthesis during germination.

(a–d) S. coelicolor wild type. (e–j) SCO4439::Tn5062 mutant. (k–n), (SCO4439::Tn5062[pBRB3] complemented strain. GYM liquid cultures were stained with BODIPY-vancomycin, and observed at the confocal microscope. The interference contrast mode (left pictures) and fluorescent images (right pictures) are shown. Arrows indicate nascent PG. Developmental time points are indicated. Scale bars represent 4 μm.

Figure 7. Model for the biological function of SCO4439.

(a) S. coelicolor wild type. (b) SCO4439::Tn5062. Classical germination stages: darkening, swelling and germ tube emergence. The proposed spore deswelling stage is indicated. Green illustrates the membrane-intact cells (SYTO 9 staining); red indicates propidium iodide (PI) permeable cells (lysis). D-iGlx, D-iso-glutamine or D-iso-glutamic acid. See text for details.