Downloaded from www.microbiologyresearch.org by IP: 54.147.215.157 On: Thu, 08 Dec 2016 22:47:27 Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis Zhiqiang Qin, 1 3 Yuanzhu Ou, 1 3 Liang Yang, 2 Yuli Zhu, 1 Tim Tolker-Nielsen, 2 Soeren Molin 2 and Di Qu 1 Correspondence Soeren Molin sm@biocentrum.dtu.dk 1 Key Laboratory of Medical Molecular Virology of Ministry of Education and Public Health, Institutes of Biomedical Sciences and Medical Microbiology, Shanghai Medical School of Fudan University Box 228, Yi Xue Yuan Road 138 # , Shanghai 200032, P. R. China 2 BioCentrum-DTU, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark Received 14 January 2007 Revised 7 February 2007 Accepted 9 February 2007 Staphylococcus epidermidis has become a serious nosocomial pathogen frequently causing infections associated with implanted foreign materials. Biofilm formation is considered a major factor determining S. epidermidis pathogenicity in such device-associated infections. Here, evidence is presented that extracellular DNA is important for the initial phase of biofilm development by S. epidermidis on polystyrene or glass surfaces under static or hydrodynamic conditions. Comparative PCR amplification from S. epidermidis chromosomal and extracellular DNA indicated that the extracellular DNA is similar to chromosomal DNA. Experiments involving the S. epidermidis wild-type and an isogenic atlE mutant indicated that most of the extracellular DNA in S. epidermidis cultures and biofilms is generated through activity of the autolysin AtlE. The presented results suggest that extracellular DNA is generated in S. epidermidis populations through AtlE-mediated lysis of a subpopulation of the bacteria, and that the extracellular DNA promotes biofilm formation of the remaining population. INTRODUCTION Bacteria in natural environments predominantly live in biofilms, i.e. surface-attached microbial communities em- bedded in a self-produced extracellular matrix (Costerton et al., 1995). The extracellular polymeric substance (EPS) matrix, which can constitute up to 90 % of the biofilm biomass (Flemming et al., 2000), is a complex mixture of exopolysaccharides, proteins, DNA and other macromole- cules (Sutherland, 2001). The EPS matrix plays a structure- stabilizing role in biofilms, and it provides protection against some antibiotics and host-defence activities (Mah & O’Toole, 2001; Stewart & Costerton, 2001). In recent years, coagulase-negative Staphylococcus epider- midis has become a serious nosocomial pathogen, fre- quently causing infections associated with implanted foreign materials (Rupp & Archer, 1994). Biofilm forma- tion is a major factor determining S. epidermidis patho- genicity in such device-associated infections (Rupp et al., 2001). S. epidermidis biofilm formation has been described as a two-step process (Gotz et al., 2000). The first stage involves attachment of cells to a surface (initial attachment phase). The second stage includes cell–cell aggregation and the formation of a multilayered architecture (accumulative phase). Much attention has been focused on the poly- saccharide intercellular adhesion (PIA) component of the EPS matrix of S. epidermidis, which is considered a major cell-to-cell interconnecting compound during biofilm for- mation (Heilmann et al., 1996a; Mack, 1999). However, it is possible that other matrix components may be important for biofilm development of S. epidermidis, such as extra- cellular DNA, which has been shown to be important for biofilm formation of Pseudomonas aeruginosa, Streptococcus intermedius and Streptococcus mutans (Whitchurch et al., 2002; Nemoto et al., 2003; Petersen et al., 2004, 2005; Allesen-Holm et al., 2006). In P. aeruginosa biofilms, extra- cellular DNA functions as a cell–cell interconnecting compound and structural component, and its production has been shown to be regulated via quorum sensing (Allesen-Holm et al., 2006). In this study, we show that extracellular DNA is present in cultures and biofilms of S. epidermidis strains under different growth conditions. The extracellular DNA is found as a major component required for initial bacterial attachment to surfaces, as well as for the subsequent early phase of biofilm development by S. epidermidis. Moreover, evidence is presented that release of extracellular DNA from S. epidermidis is mainly caused by the activity of the autolysin AtlE. Abbreviations: CLSM, confocal laser scanning microscopy; DDAO, 7- hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one); EPS, extracellular polymeric substance; PI, propidium iodide. 3These authors contributed equally to this work. Microbiology (2007), 153, 2083–2092 DOI 10.1099/mic.0.2007/006031-0 2007/006031 G 2007 SGM Printed in Great Britain 2083