Ultrastructural and electron energy-loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate

Strain F8, a bacterial isolate from 'river snow', was found to produce extracellular fibres in the form of a filamentous network. These extracellular filaments, which were previously shown to be composed of DNA, have been studied for the first time by ultrastructural and electron energy-loss spectroscopy in the present work. 'Whole mount' preparations of strain F8 indicate these polymers are ultrastructurally homogeneous and form a network of elemental filaments, which have a width of 1.8-2.0 nm. When incubated at pH 3.5 with colloidal cationic ThO2 tracers they become intensely stained (electron dense), affording direct evidence that the fibres are negatively charged and thus acidic chemically. Elemental analysis of the extracellular filaments by Energy-filtered Transmission Electron Microscopy revealed phosphorus to be the main element present and, because pretreatment of F8 cells with DNase prevented thorium labelling, the fibres must be composed of extracellular DNA (eDNA). Neither ultrathin sections nor 'whole mount negative stain' caused DNA release by general cell lysis. Additionally, cells infected with phages were never observed in ultrathin sections and phage particles were never detected in whole mount samples, which rules out the possibility of phages being directly involved in eDNA release.