Bacterial extracellular DNAforming a de¢ned network-like structure Uta B ¨ ockelmann 1 , Andrea Janke 1 , Ramona Kuhn 1 , Thomas R. Neu 2 ,J¨ org Wecke 3 , John R. Lawrence 4 & Ulrich Szewzyk 1 1 Department of Environmental Microbiology, University of Technology Berlin, Berlin, Germany; 2 Department of River Ecology, UFZ Centre for Environmental Research Leipzig-Halle, Magdeburg, Germany; 3 Robert Koch Institute, Berlin, Germany; and 4 National Water Research Institute, Saskatoon, Saskatchewan, Canada Correspondence: Uta B ¨ ockelmann, Technical University Berlin, Environmental Microbiology, Franklinstrasse 29, FR 1-2, 10587 Berlin, Germany. Tel.: 149 3031473566; fax: 149 3031473673; e-mail: uta.boeckelmann@tu-berlin.de Received 28 January 2006; revised 28 April 2006; accepted 9 June 2006. First published online 10 July 2006. DOI:10.1111/j.1574-6968.2006.00361.x Editor: Sylvie Rimsky Keywords extracellular DNA (eDNA); filamentous network; aquatic bacteria; nanotechnology; nanostructures. Abstract It is generally assumed that nucleic acids are localized inside of living cells and that their primary function is the storage of information. In contrast, extracellular DNA is mainly considered as a remnant of lysed cells. Here, we report the formation of extracellular bacterial DNA as a spatial structure. An aquatic bacterium, strain F8, was isolated, which produced a stable filamentous network of extracellular DNA. Different staining and enzymatic techniques confirmed that it was DNA. We were able to amplify the 16S rRNA gene from the extracellular DNA. Restriction endonuclease cleavage and randomly amplified polymorphic DNA analysis of extracellular and genomic DNAs revealed major similarities, but also some differences in both sequences. Our data demonstrate a new function and relevance for extracellular DNA. Introduction Bacteria predominantly live in communities, such as biofilms or bioaggregates in which extracellular polymeric substances (EPS) form the matrix and stabilize the spatial structure (Watnick & Kolter, 2002). DNA has not been considered as an important EPS component but rather as originating from lysed cells. Studies on the degradation and turnover of extracellular DNA (eDNA) in marine sediments suggest their role in the P cycle of marine ecosystems (Dell’Anno & Corinaldesi, 2004). eDNA concentrations in aquatic sedi- ments were three to four orders of magnitude greater than those in the water column (Corinaldesi et al ., 2005). Conse- quently, Dell’Anno & Danovaro (2005) stated that eDNA plays a key role in the functioning of deep-sea ecosystems. It is known that some bacteria produce substantial quantities of eDNA. Olishevsky et al. (2004) discovered quantities of unmethylated CpG in the eDNA of two Bacillus subtilis strains. Moreover, it was shown that Pseudomonas aeruginosa produces a large amount of eDNA during alginate biosynthesis, which is clearly required for the initial biofilm establishment of the strain (Steinberger et al., 2002; Whitchurch et al., 2002). Steinberger & Holden (2005) recently investigated the universality and persistence of eDNA in dual-species biofilms. It was also reported that the flocculation ability of a marine photosynthetic Rhodovulum sp. was due to secreted nucleic acids (Watanabe et al., 1998). The characterization of these nucleic acids revealed a mixture of double-stranded DNAs and single-stranded RNAs (Nishimura et al., 2003). Whereas all these studies dealt with eDNA as an unstruc- tured part of the EPS, in this paper, we describe for the first time eDNA as a major structural component of the EPS of a defined pure culture. Materials and methods Isolation of strain F8 In order to characterize the microbial community, with emphasis on oligotrophic bacteria of lotic organic aggre- gates, bacteria from ‘river snow’ of the South Saskatchewan River were isolated using oligotrophic freshwater basal medium (FBM). It contained 3.0 g Na 2 SO 4 , 0.4 g MgCl 2  6H 2 O, 1.2 g NaCl, 0.3 g NH 4 Cl, and 0.15 g CaCl 2  2H 2 O, and was supplemented with yeast extract (0.01% w/v) plus glucose (0.5 mM) trace element and vitamin solution (B¨ ockelmann et al., 2000). Serial dilutions FEMS Microbiol Lett 262 (2006) 31–38 c  2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsle/article/262/1/31/477328 by guest on 16 June 2022