Extracellular DNA (eDNA) is a structural component of the polymeric matrix of biofilms from different species. Different mechanisms for DNA release have been proposed including lysis of cells, lysis of DNA-containing vesicles, and DNA secretion. Here, a genome-wide screen of 3985 non-lethal mutations was performed to identify genes whose deletion alters eDNA release in Escherichia coli. Deleting nip!, yfeC, and rna increased eDNA from planktonic cultures while deleting hns and rfaD decreased eDNA production. The lipoprotein NlpI negatively affects eDNA release since the overexpression of nlpI decreases eDNA 16 fold while deleting nlpI increases eDNA threefold. The global regulator H-NS is required for eDNA production since DNA was not detected for the hiss mutant and production of H-NS restored eDNA production to wild-type levels. Therefore our results suggest that secretion may play a role in eDNA release in E. coli since the effect of the hiss deletion on cell lysis (slight decrease) and membrane vesicles (threefold increase) does not account for the reduction in eDNA. (C) 2010 Elsevier Inc. All rights reserved.