Investigating Extracellular DNA Release in Staphylococcus xylosus Biofilm In Vitro

Abstract

Staphylococcus xylosus forms biofilm embedded in an extracellular polymeric matrix. As extracellular DNA (eDNA) resulting from cell lysis has been found in several staphylococcal biofilms, we investigated S. xylosus biofilm in vitro by a microscopic approach and identified the mechanisms involved in cell lysis by a transcriptomic approach. Confocal laser scanning microscopy (CLSM) analyses of the biofilms, together with DNA staining and DNase treatment, revealed that eDNA constituted an important component of the matrix. This eDNA resulted from cell lysis by two mechanisms, overexpression of phage-related genes and of cidABC encoding a holin protein that is an effector of murein hydrolase activity. This lysis might furnish nutrients for the remaining cells as highlighted by genes overexpressed in nucleotide salvage, in amino sugar catabolism and in inorganic ion transports. Several genes involved in DNA/RNA repair and genes encoding proteases and chaperones involved in protein turnover were up-regulated. Furthermore, S. xylosus perceived osmotic and oxidative stresses and responded by up-regulating genes involved in osmoprotectant synthesis and in detoxification. This study provides new insight into the physiology of S. xylosus in biofilm.

Keywords: DNA/RNA repair; Staphylococcus xylosus; amino sugar catabolism; biofilm; cell lysis; eDNA; protein turnover.

Figure 1

3D projection of biofilms of S. xylosus C2a-B2 and DNA stained by TOTO-3 (A), after DNase treatment at the end of each incubation time (B), after incubation in the presence of DNase from the start (C), at three sampling times using Imaris software; Scale bar—30 µm.

Figure 2

Quantitative structural parameters of the biofilms of S. xylosus C2a-B2 and eDNA stained by TOTO-3 at three sampling times. (A) Biovolume, (B) Coverage, (C) Average thickness and (D) roughness coefficient. Biovolume C (µm³) represented the overall volume of cells in the observation field. Coverage (%) reflected the efficiency of substratum colonization by bacteria. The average thickness (μm) of biofilms was determined from the confocal stack images. Roughness coefficient provided a measure of variations in biofilm thickness and was an indicator of the superficial biofilm interface heterogeneity. Three independent experiments were performed per sampling time. C2a-B2 control (continuous blue curve) and eDNA control (continuous red curve): no treatment; C2a-B2 + DNase t (dashed blue curve) and eDNA + DNase t (dashed red curve): DNase treatment at the end of each incubation time; C2a-B2 + DNase to (dotted blue curve) and eDNA + DNase to (dotted red curve): after incubation in the presence of DNase from the start.

Figure 3

Transmission electron microscopy of phages in the supernatant of S. xylosus after induction with mitomycin C. Bar, 100 nm. Magnification, ×120,000.

Figure 4

Summary of carbohydrate and amino sugar catabolism in S. xylosus biofilm showing the up-regulated genes (the level of expression of these genes and the corresponding enzymes are given in Table 1). GlcNac-6P = N-acyl-d-glucosamine 6-phosphate, ManNac = N-acyl-d-mannosamine, ManNac-6P = N-acyl-d-mannosamine 6-phosphate, Neu5Ac = N-acetyl neuraminate.