Extracellular DNA (exDNA) is abundant in many habitats, including soil, sediments, oceans and freshwater as well as the intercellular milieu of metazoa. For a long time, its origin has been assumed to be mainly lysed cells. Nowadays, research is collecting evidence that exDNA is often secreted actively and is used to perform a number of tasks, thereby offering an attractive target or tool for biotechnological, medical, environmental and general microbiological applications. The present review gives an overview on the main research areas dealing with exDNA, depicts its inherent origins and functions and deduces the potential of existing and emerging exDNA-based applications. Furthermore, it provides an overview on existing extraction methods and indicates common pitfalls that should be avoided whilst working with exDNA. . . . . . Keywords Extracellular DNA Environment Biofilm Soil Plant Microbial activity Introduction give the preference to the acronym exDNA. Marine biologists often differentiate between aqueous-extractable Bsoluble In contrast to intracellular DNA (iDNA), which is the DNA DNA^ (sDNA) and Bnon-soluble DNA^ (nsDNA); both frac- located within cell membranes, extracellular DNA (exDNA) tions are roughly representing exDNA and iDNA, respective- represents the DNA located outside thereof. Such DNA can be ly (Lever et al. 2015). The acronyms esDNA, aDNA and found in any kind of environmental samples. One of the best cirDNA stand for extracellular self-DNA, ancient DNA and definitions including information about its origin was given circulating DNA, respectively, and will be addressed in the by Pietramellara et al. (2009), stating that exDNA is chapters Bsoil^, Bmarine and lake ecosystems^ and Bhuman Boriginating from intracellular DNA by active or passive ex- body^,respectively. trusion mechanisms or by cell lysis^. When it has become known to be common in the environ- Dealing with environmental DNA, several abbreviations ment in the early 1950s, exDNA was studied in the context of are used to refer to similar or different items. Whilst a common horizontal gene transfer (HGT) (Avery et al. 1944; Freeman acronym for environmental DNA is eDNA, a number of au- 1951) and the ability of microorganisms to achieve antibiotic thors used this acronym for extracellular DNA, too. resistance through transformation by foreign (extracellular Additionally, the terms exDNA or cfDNA (cell-free DNA) plasmid) DNA (Akiba et al. 1960; Romanowski et al. 1993). were introduced to refer to extracellular DNA in order to pre- During the 80s and 90s of the past century, exDNA was stud- vent confusion with environmental DNA. In this review, we ied in terms of its persistence in soil, i.e. protection against nuclease degradation due to binding to various soil compo- nents (Ogram et al. 1987; Paget et al. 1992; Vettori et al. 1996), and its degradation rates in estuarine and marine envi- * Magdalena Nagler ronments (Paul et al. 1987). firstname.lastname@example.org Frostegård et al. (1999) evaluated DNA extraction efficien- cies of several protocols and addressed the issue of extracting Universität Innsbruck, Institute of Microbiology, Technikerstr. 25d, extracellular and intracellular soil DNA simultaneously. By 6020 Innsbruck, Austria then, exDNA was found to be omnipresent, and with this Dipartimento di Scienze delle Produzioni Agroalimentari e awareness, a variety of research foci on different natural envi- dell’Ambiente, Università degli Studi di Firenze, Piazzale delle Cascine 18, 50144 Florence, Italy ronments emerged: 6344 Appl Microbiol Biotechnol (2018) 102:6343–6356 & The persistence and ecological relevance of exDNA in soil concentrations, low pH and a high content of expandable clay (reviewed by Levy-Booth et al. 2007; Pietramellara et al. minerals have all been found to slow down exDNA degrada- 2009); tion (Crecchio et al. 2005; Pietramellara et al. 2009). An at- & The persistence, function and turnover of exDNA in ma- tempt to estimate the age of soil exDNA by radiocarbon dating rine and aquatic ecosystems (reviewed by Torti et al. suggested a survival time ranging from 21,000 years ( Cage) 2015); to 900,000 years (mean residence time), even though it was & The occurrence, relevance of exDNA and possible suggested to treat these results with care, as a contamination exDNA-derived therapies in the human body (reviewed (e.g. with fossil carbon) could not be totally excluded (Agnelli by Aucamp et al. 2016;Cooper etal. 2013; Thierry et al. et al. 2007). Despite its binding to various minerals, exDNA 2016); still preserves its ability to transform competent microbial & The importance and the functions of exDNA in the forma- cells in the soil (Fig. 1)(Morrissey et al. 2015;Romanowski tion of biofilms of pathogenic and environmental micro- et al. 1993; Thomas and Nielsen 2005). Whilst some studies organisms (reviewed by Montanaro et al. 2011 and suggested that HGT frequencies in soil are low (Nielsen et al. Wnorowska et al. 2015 (exDNA), Hobley et al. 2015 1998; Pietramellara et al. 2007; Pietramellara et al. 2006; (biofilms in general), Wolska et al. 2016 (genetic control), Thomas and Nielsen 2005), some hypothesised that the actual Payne and Boles 2016 (matrix interactions and resulting transformation rates are underestimated due to the high num- implications) and Azeredo et al. 2017 (methods)). ber of unculturable microorganisms (Pietramellara et al. 2009). However, the long persistence of DNA in soil brings Extracellular DNA has also been investigated within dead about an increased presence of antibiotic resistance genes that wood (Gómez-Brandón et al. 2017a), cattle rumen and ma- might be passed from cell to cell (Poté et al. 2003), with both nure (Chroňáková et al. 2013; Fliegerová et al. 2014; Nagler et ecological and evolutionary implications. The quality of al. 2018), aerobic and anammox granules (Cheng et al. 2011; exDNA is depending on its state of degradation, fragment Xiong and Liu 2012;Dominiak et al. 2011) and human epi- sizes ranging from 80 to more than 20,000 bp, as shown by thelial cells used in forensics (Wang et al. 2017). In addition, standard agarose gel electrophoresis (Ascher et al. 2009b). exDNA was found to act as a trap for infectious organisms in The integrity of large fragments of exDNA was shown by mammalians (reviewed by Ciesluk et al. 2017) and during root the successful amplification of a 1700-bp portion, almost the tip growthofplants(Hawesetal. 2012; Pietramellara et al. complete fungal 18S gene (Ascher et al. 2009b). A large per- 2013). Finally, exDNA is assumed to act as a species-specific centage of exDNA in soil was found to be double stranded, growth inhibitor all over the tree of life (Mazzoleni et al. being detectable with methods specifically binding to double- 2015b;esDNA). stranded DNA (intercalation dyes, e.g. PicoGreen) (Agnelli et Whilst most of recently published reviews regarding al. 2004;Ascher et al. 2009b). exDNA focus on a single specific environment, the present After active or passive excretion or release from lysed cells review aims to summarise the main features, functions and (i.e. after cell death/necrosis or virus attack), exDNA can be pertinences of exDNA in all so far investigated natural envi- diffused in the soil through various mechanisms. Vertically, ronments (Fig. 1). In doing so, we also intend to depict the movement was found to be either directed towards the existing as well as emerging exDNA-based applications. groundwater through leaching or towards the soil surface Furthermore, we give a short overview on existing extraction through advection in water capillaries; horizontally, move- methods and indicate common pitfalls that should be avoided ment follows the soil water flow direction (Agnelli et al. whilst working with exDNA. 2004;Ascher etal. 2009a; Ceccherini et al. 2007;Poté etal. 2003). In both directions, exDNA may reach areas with little nutrient content. Accounting for over 10% of the extractable P Soil in soil and containing essential elements such as N and C, exDNA may act as a nutrient and energy source especially In soils, exDNA is omnipresent and has first been studied with in soils with low nutrient input (reviewed by Levy-Booth et regard to its adsorption to sand, clay and other soil colloids al. 2007; Nielsen et al. 2007). After a breakdown by extracel- (Fig. 1) (e.g. Lorenz and Wackernagel 1987; Paget et al. 1992; lular and cell-associated nucleases (DNases), smaller exDNA Pedreira-Segade et al. 2018). Once bound to these particles, molecules are taken up by microbial cells, where they either exDNA is partly physically protected from degradation, serve as building blocks for newly synthetized nucleic acids or allowing persistence for years (Agnelli et al. 2007;Nielsen are further broken down to essential nutrients (Morrissey et al. et al. 2007). The actual persistence of exDNA depends on a 2015). number of factors such as its composition, methylation or Just like in other environments, soil exDNA plays a crucial conformation and the prevailing environmental conditions. role in the formation of biofilms, exhibiting mainly structural functions as discussed below and serves as an information In that context, rapid desiccation, low temperatures, high salt Appl Microbiol Biotechnol (2018) 102:6343–6356 6345 Fig. 1 Main functions of extracellular DNA (exDNA) in different natural environments. Darker shaded areas represent functions deriving from the informational character of exDNA, whilst lighter areas comprise functions owed to the Bsticky^ character of exDNA pool for HGT. Similarly, soil particles and organisms such as one hand and protection from desiccation and predation in a microalgae and microorganisms are known to form biological low-potential activity regime on the other (Young and soil crusts particularly in the topsoil of arid soils, where the Crawford 2004). Supporting the formation of pores and ag- production of extracellular polymeric substances (EPS) in- gregates according to its structural properties, exDNA could cluding exDNA leads to an increased water retention (e.g. possibly contribute to this self-organisation. Adessi et al. 2018). Such soil-microbe systems are thought Bearing additional taxonomic and phylogenetic informa- to be self-organised in a way that microbes shape the state of tion with regard to iDNA, exDNA has therefore been used oxygen supply through their activity (respiration), causing a to compare information about microbial communities deriv- shift between oxygen supply and high potential activity on the ing from both fractions of the total soil DNA pool (Agnelli et 6346 Appl Microbiol Biotechnol (2018) 102:6343–6356 al. 2004;Ascheretal. 2009b;Ceccherinietal. 2009; has the function of a signalling compound. In the context of Chroňáková et al. 2013;Gómez-Brandónetal. 2017b). root growth itself, its role is different. Wen et al. (2009)re- These studies revealed that some sequences found in the ported that exDNA is a component of the root cap slime exDNA fraction are not found in the iDNA fraction of the known to be involved in the increased resistance of growing total DNA pool and suggest that they are ancient or so- root caps against soil-borne pathogens, and that exDNA deg- called relic DNA. Such DNA, potentially persisting in soil radation resulted in a loss thereof (Wen et al. 2009). Later on, for long time spans, reflects the historical biodiversity of the several studies suggested that exDNA actively exported from investigated environment and can give important information the root tip may function similar to the exDNA secreted in about past climatic conditions (see the BApplications^ sec- human neutrophil extracellular traps (NETs) and traps patho- tion). A study conducted by Carini et al. (2016) actually genic microorganisms in close proximity to the root tips showed that the exDNA inflated the observed prokaryotic (reviewed by Hawes et al. 2011): once released by active and fungal richness by up to 55% if compared to iDNA only. secretion(Wenetal. 2017), the exDNA attracts and Following these findings, it was argued that the quantitatively immobilises pathogens as well as soil contaminants in a relevant presence of exDNA might also cause an underesti- host-microbe specific manner (Hawes et al. 2012; Hawes et mation of the actual temporal and spatial variability of soil al. 2016; Pietramellara et al. 2013). microbial communities (Fierer 2017). This may put a new Not strictly soil but still closely related, antimicrobial resis- perspective on the concept of Beverything is everywhere, but tance might emerge with increased frequency in livestock the environment selects^, stating that most species are present waste management structures. Zhang et al. (2013) found that at least in low abundances in all soils and will thrive as soon as several antimicrobial resistance genes were present in the the environmental conditions allow for (Baas Becking 1931; exDNA and iDNA pool of such environments and that HGT Fenchel and Finlay 2004;Nagler et al. 2016). For any assump- is a potential mechanism for the spread of antimicrobial resis- tions concerning diversity and microbial species abundance, it tance. Investigating rumen-borne microbial communities, is thus indispensable to distinguish between environmental considerable differences between exDNA and iDNA bacterial DNA (eDNA) and exDNA on the one hand, and the extracel- profiles have been found (Fliegerová et al. 2014), suggesting lular (exDNA) and intracellular fraction (iDNA) of the total differing lysis and/or DNA secretion of the microorganisms. DNA pool on the other (reviewed by Taberlet et al. 2012a) (see the BApplications^ section). In an investigation on litter autotoxicity, the role of ex- Marine and aquatic ecosystems tracellular self-DNA (esDNA) has first been addressed by Mazzoleni et al. (2015a), who found that the growth not In the marine environment, exDNA is present throughout, only of plants but also of soil animals and microorganisms from the estuarine to the anoxic deep sea. Its origin, dynamics was inhibited when conspecific exDNA was added to the and implications have been reviewed by Torti et al. (2015). It growth substrate (Mazzoleni et al. 2015a, b). This effect is estimated that around 90% of the total DNA pool in the was found to be very specific and applied only for conspe- ocean occur as exDNA (Dell'Anno and Danovaro 2005), cific but not for other heterologous exDNA. The authors which accounts for a global 0.45 Gt of DNA in the uppermost hypothesised that this inhibition effect represents a mech- 10 cm of sea water, where amounts of exDNA are three orders anism of maintaining diversity. In an attempt to interpret of magnitudes lower than in sediments (Torti et al. 2015). these far-reaching findings, Veresoglou et al. (2015) Marine exDNA is either autochthonous or allochthonous, pas- discussed that esDNA in soil could function as a conspe- sively or actively released from decaying, virus-attacked or cific stress-signalling molecule rather than an inhibitory growing (micro)organisms. If the exDNA is released in the substrate. Similarly, Duran-Flores and Heil (2015) argued water column, it sediments only if complexed with particles that esDNA could belong to the group of damage- heavy enough to sink to the sea floor (Herndl and Reinthaler associated molecular patterns (DAMP) that cause the local 2013). However, once released, the fate of exDNA includes development of resistance-related responses by the affect- natural transformation, degradation through ubiquitous ed plant. All these findings, however, are rather prelimi- DNases and subsequent incorporation by microbial cells, nary and require additional research to adequately interpret long-term preservation and abiotic decay (Fig. 1). As for and describe the underlying mechanisms. long-term preservation, binding of exDNA in marine sedi- Finally, the role of exDNA in soil is also linked to plant ments is similar to that of soil; the interaction is electrostatic physiology. The presence of exDNA in the growth medium of and requires the presence of inorganic cations to bind the plants enhances the growth of lateral roots and root hairs and negatively charged inorganic and organic sediment surfaces the effect is linked to an altered expression of specific peptide with the phosphate groups of DNA (Fig. 1)(Lorenz and hormone genes that are controlling root morphology Wackernagel 1987). Furthermore, exDNA is preserved after (Paungfoo-Lonhienne et al. 2010). In that context, exDNA contact with brines of deep anoxic hypersaline lakes (Borin et Appl Microbiol Biotechnol (2018) 102:6343–6356 6347 al. 2008), where non-adapted bacteria might lyse with a higher organised in clear patterns, forming grid-like structures or fil- frequency due to osmotic stress, giving rise to an environment amentous networks (Fig. 1) (Allesen-Holm et al. 2006; favouring high rates of HGT. Böckelmann et al. 2006;Flemming etal. 2007). As a conse- Next to exDNA in the water column and in the sediments, quence, exDNA has been described as a structural component exDNA can also be located in the extracellular polymeric of the extracellular matrix, being essential especially during substance (EPS) of marine biofilms, as reviewed by Decho biofilm formation (Conover et al. 2011; Kawarai et al. 2016; and Gutierrez (2017). EPS form a major component of the Martins et al. 2010; Novotny et al. 2013; Nur et al. 2013; total pool of dissolved organic carbon in the ocean, but the Seper et al. 2011;Whitchurch et al. 2002;Zhao etal. 2013) role of exDNA in this specific environment has not been in- (reviewed by Flemming et al. 2016; Montanaro et al. 2011) vestigated so far. and thus being actively secreted by the biofilm-producing mi- Regarding lake and other freshwater environments, croorganisms (Barnes et al. 2012; Kilic et al. 2017;Liaoet al. exDNA-related studies are very scarce. A study reporting 2014; Rose and Bermudez 2016; Zafra et al. 2012). A about ferruginous sediments in a tropical lake in Indonesia genome-wide screening for genes involved in exDNA release used the exDNA bound to the sediment to study the microbial during biofilm formation by S. aureus was recently done consortium and detected exDNA in decreasing amounts from (DeFrancesco et al. 2017). the lake ground to 30-cm sediment depth as well as differences In biofilms of mixed bacterial consortia such as granular in the taxonomic composition between exDNA and iDNA activated sludge, differences in the composition of exDNA vs. (Vuillemin et al. 2016). Another study focussed on the persis- iDNA were detected applying a fingerprinting approach tence of antimicrobial resistance genes in the exDNA pool of a (Cheng et al. 2011) and indicating a species-specific DNA river sediment and reported that resistance genes often incor- release originating mostly from active secretion (Dominiak porated into plasmid DNA exhibit a longer persistence than et al. 2011). Moreover, microbial aggregation during aerobic chromosomically encoded 16S rRNA genes, suggesting that granulation and consequently biomass density and size are exDNA represents a major reservoir for antibiotic resistance positively affected by increased exDNA amounts (Xiong information (Mao et al. 2014). In the Arctic sea ice, exDNA and Liu 2012). In oral biofilms, the exDNA consists not only has been found in concentrations higher than those reported of microbial but also of host-DNA but exhibits similar func- from any marine environment and it was hypothesised that sea tions than in other biofilms (reviewed by Jakubovics and ice may be a hotspot for HGT in the marine environment Burgess 2015; Schlafer et al. 2017). (Collins and Deming 2011). Focusing on the role of exDNA in biofilms, several studies (Doroshenko et al. 2014; Hathroubi et al. 2015; Schilcher et al. 2016) found increased exDNA concentrations after exposure Biofilms to low concentrations of antibiotics and vice versa, a higher antimicrobial resistance with higher amounts of exDNA One of the best-studied environments housing exDNA are (Johnson et al. 2013;Lewenza 2013), suggesting a protective biofilms, the focus lying particularly on those formed by clin- function. Through its negative charge, exDNA acts as a che- ically relevant microorganisms such as Staphylococcus spp., lator of cationic antimicrobials (Mulcahy et al. 2008) but can Streptococcus spp., Candida spp., Pseudomonas aeruginosa also act as a protection system against aminoglycosides and mixed oral biofilms. Other biofilms formed by environ- (Chiang et al. 2013). The main protective power against anti- mental microorganisms, plant pathogens (Sena-Velez et al. microbials or predation, however, is owed to the exDNA’s 2016), or in the activated sludge during wastewater treatment function to structurally stabilise biofilms and thereby increase have been studied to a lesser extent (e.g. Dominiak et al. antimicrobial resistance (see the BApplications^ section). 2011). exDNA has also been shown to attract and bind with positive- The presence of DNA in the EPS and its responsibility for ly charged amyloids in various biofilms, thereby accumulating the stickiness of the by then so called Bslime^ or Bmats^ was peptides and causing a polymerisation of the matrix and stim- discovered as early as in 1955 for some halophilic bacteria ulating autoimmunity (reviewed by Payne and Boles 2016; (Smithies and Gibbons 1955) and several years later with a Randrianjatovo-Gbalou et al. 2017; Schwartz et al. 2016). focus on human pathogens for Pseudomonas aeruginosa An interaction with polysaccharides was found in P. (Murakawa 1973). Beginning in 1996, exDNA was increas- aeruginosa and S. mutans biofilms, where both components ingly noted in the EPS matrix of activated sludge and in pure form a web of fibres and function as a skeleton allowing bac- cultures of Pseudomonas putida (reviewed by Flemming and teria to adhere and grow (Payne and Boles 2016; Pedraza et al. Wingender 2010). The origin of this DNA has long thought to 2017). be lysed cells. Later, it was found that the exDNA is present in The role of exDNA as a source of genetic information in species-specific amounts in different single- and multiple- the context of HGT within the biofilm has been addressed in several studies (e.g. Merod and Wuertz 2014; Wang et al. species biofilms (Steinberger and Holden 2005) and that it is 6348 Appl Microbiol Biotechnol (2018) 102:6343–6356 2002) and was found to occur frequently, as biofilms are synthesising DNA extracellularly. If originating, however, hotspots, i.e. offer ideal conditions for HGT including high from such an active cellular release mechanism, exDNA is cell density, increased genetic competence and an accumula- often bound to other plasma constituents such as RNA, lipids tion of exDNA. Conjugation has been shown to be up to 700- and proteins, being in that case called virtosomes (Fig. 1). As fold more efficient in biofilms compared to planktonic bacte- part of virtosomes, exDNA shows the ability to migrate to rial cells (Flemming et al. 2016), further promoting antimicro- different parts of the body, enter target cells and alter their bial resistance in biofilms. Moreover, several other functions physiological properties such as the immune response, by of exDNA in biofilms have been described. In most biofilms, sharing antigenic information (Anker et al. 1984;Aucampet exDNA is needed throughout the biofilm development al. 2016;Skogetal. 2008). Peters and Pretorius (2012) (Brockson et al. 2014) but especially for the initial adhesion highlighted that this active release and uptake of nucleic acids and aggregation of bacteria on surfaces (Das et al. 2010;Das is a characteristic of all organisms and cell types, and that in et al. 2011;Jermy 2010;Tanget al. 2013). In Caulobacter contrast to the neo-Darwinian dogma, physical and behaviour- crescentus biofilms, however, exDNA binds to the holdfast al traits can be inherited through this cycling. This is because of swarmer cells, promotes their dispersal to places with less there has been found evidence that not only somatic but also present exDNA and thereby prevents biofilm maturation germ cells might be subject to genetic and epigenetic modifi- (Berne et al. 2010; Kirkpatrick and Viollier 2010). cations via exDNA (intensively reviewed and discussed by Furthermore, it has been suggested that self-organisation of Aucamp et al. 2016). In this context, it has been hypothesised cells in actively expanding biofilms of P. aeruginosa occurs that the exDNA in human blood vessels might derive to a directly on the exDNA filaments (Böckelmann et al. 2006)or large extend from metabolic DNA, which is—as opposite to through the construction of a network of furrows supported by thestablegeneticDNA—a specially synthesised low- exDNA molecules (Gloag et al. 2013). During mechanical molecular-weight fraction of DNA involved in the regulation stress of a biofilm, exDNA was found to exhibit a distinguish- and performance of RNA production and other cellular func- able role in controlling the viscoelastic relaxation of the bio- tions. Deriving from such a de novo synthesis in cells (van der film (Peterson et al. 2013). In addition, Sapaar et al. (2014) Vaart and Pretorius 2008), exDNA differs from the DNA in suggested that exDNA may induce the morphological change the nucleus containing single- and double-strain breaks and from yeast to hyphal growth in C. albicans biofilms, but with- accumulations in GC-rich regions (Veiko et al. 2008). out providing any explanation about the possible underlying Another field of studies regarding exDNA in the human mechanisms. body is the immune system, where neutrophils secrete exDNA together with actin, histone, peroxidases and proteins, thereby forming a neutrophil extracellular trap (NET), a sticky matrix around the cell (Fig. 1)(Brinkmann et al. 2004). These Human body NETs are part of the immune system and are formed as a Next to the exDNA secreted by clinically relevant microor- response to defence-pathway-inducing signals. Pathogens ganisms forming biofilms on or inside the human body (cov- can be chemotactically attracted by the NETs and are then ered in the section biofilm), exDNA of predominantly endog- immobilised and potentially killed by the antimicrobial com- enous origin can be found in the extracellular milieu of the ponents of the trap (Halverson et al. 2015; Hawes et al. 2015). human body including blood, lymph, bile, milk, urine, saliva, Hawes et al. (2015) proposed that NETs gain most of their mucous suspension, spinal and amniotic fluid. Beginning in bactericidal character through the removal of surface- the 1960s, exDNAwas discovered in the plasma and serum of stabilising bacterial cations by the DNA phosphodiester back- patients with a variety of diseases, including rheumatoid ar- bone, resulting in bacterial lysis. Recent studies, however, thritis, pancreatitis, inflammatory bowel disease, hepatitis and revealed that an overproduction of NETs followed by an ac- oesophagitis. By the 1970s, it was shown to be double- cumulation of exDNA contributes to the pathogenesis of some stranded and of a similar size range as in soil (i.e. from 180 diseases. Breast cancer cells can induce neutrophils to produce to 10,000 bp) (van der Vaart and Pretorius 2008). With the NETs without infection (Park et al. 2016), thereby exploiting development of more sensitive assays, it was found to be also the host cells in order to promote metastases. Furthermore, present in healthy subjects, albeit to a lesser extent (Anker et NETs can cause the aggregation and implantation of cancer al. 1999). It has been proposed that this type of circulating cells due to its sticky character (Hawes et al. 2015). In this exDNA (cirDNA, cell-free cfDNA or plasma DNA) is re- context, the genometastasis hypothesis was formulated, stat- leased by apoptosis and necrosis, by bacteria and viruses, ing that exDNA derived from tumour cells just like virtosomes and via active release from highly proliferating cells can enter healthy cells and lead to the formation of metastases (reviewed by Thierry et al. 2016). Anker et al. (1976) obtained as reviewed by García-Olmo et al. (2012) and discussed by evidence that human lymphocytes can release complexes con- Thierry et al. (2016). A variety of other pathologies have re- taining DNA or produce enzymes that are capable of cently been linked to exDNA, including the chronic airway Appl Microbiol Biotechnol (2018) 102:6343–6356 6349 disease, where NETs accumulate in the airways leading to an In biofilm research, exDNA extraction without contamina- activation of the innate immune system and impairing the tion of genomic DNA was found to work best with enzymatic patients’ state of health (Wright et al. 2016). Similarly, in treatment methods yielding more exDNA than a simple cen- patients suffering from dry eye disease, the production and trifugation (Wu and Xi 2009). In cancer research, a discrimi- degradation of exDNA is altered, allowing exDNA and nation between weakly bound and tightly bound exDNA is NETs to accumulate in the tear film and resulting in an inflam- made, and accordingly, a first step to remove weakly bound mation (Sonawane et al. 2012; Tibrewal et al. 2013). exDNA is applied using 5 mM EDTA and a second step using On the one hand, the functional role of exDNA inside the trypsin to remove exDNA tightly bound to cell surfaces is human body and especially the blood vessels is to serve as an suggested (Laktionov et al. 2004). intercellular messenger in the shape of virtosomes (Gahan and In general, independent of the environmental matrix, any Stroun 2010), spreading the immunological information about harsh step (physico-chemical) has to be avoided during the pathogenic invaders but also supporting the dissemination of extraction procedure, so as to avoid potential cell lysis. malignant information causing oncogenesis, cell invasion, metastasis and the development of resistance against radio- therapy and chemotherapy (Aucamp et al. 2016). On the other Applications hand, exDNA has been shown to act as a trap for invading pathogens in the shape of NETs, being in that way a part of the exDNA as source of specific genetic information innate immune system and combatting an infection. The same benign NETs can cause, however, pathophysiological effects One of the most immanent features of exDNA is the addi- in cancer, autoimmune pathologies, sepsis, thrombotic illness tional phylogenetic information with respect to iDNA. and in the inflammatory response through different mecha- Therefore, exDNA can be used to improve the accuracy of nisms, as highlighted above (Ciesluk et al. 2017; Cooper et assessing the soil microbial community composition al. 2013;Park etal. 2016). (Pietramellara et al. 2009), e.g. via comparative genetic fin- gerprinting of the extracellular and intracellular fraction of the total DNA pool (Agnelli et al. 2004; Ascher et al. 2009b; Methodological considerations with exDNA Chroňáková et al. 2013) or via quantitative PCR (Gómez- extraction Brandón et al. 2017a, b). Extraction methods targeting exDNA vary amongst environ- exDNA as a proxy of microbial activity (microbial mental matrices. In the soil, exDNA can strongly be bound to turnover) soil colloids like clay minerals or humic acids, resulting in a co-extraction of organic and inorganic soil compounds inter- Another feature is the origin of exDNA in various environ- fering with downstream analyses. To overcome these prob- ments, which was expected to be mainly lysed (dead) cells lems and prevent a lysis of intact cells, exDNA is desorbed (Levy-Booth et al. 2007), whilst iDNA is attributed to intact from soil particles via slightly alkaline solutions or phosphate (alive and potentially alive) cells. Consequently, a ratio of both buffers and yielded in the supernatant after centrifugation, DNA fractions (exDNA:iDNA) might provide a reliable ap- avoiding the use of cell-lysing reagents and optionally includ- proximate measure for microbial activity in soils and other ing DNase inhibitors (e.g. Agnelli et al. 2007;Ascher et al. environments (Gómez-Brandón et al. 2017a, b; Nagler et al. 2009b; Ceccherini et al. 2009; Ogram et al. 1987; Taberlet et 2018). Surprisingly, the activity of different microbes was al. 2012b). Applying such a sequential extraction, next to found to not correlate perfectly with the ratio of providing exDNA, increases the total amount of not only ex- exDNA:iDNA but could best be tracked measuring exDNA tractable soil DNA but also that of iDNA (e.g. Ascher et al. amounts without relation to iDNA (Nagler et al. 2018). These 2009b;Nagler et al. 2018; Wagner et al. 2008). results suggested that exDNA is released by microorganisms Analogously, a discrimination between sDNA and nsDNA proportional to their activity. Similarly, Dlott et al. (2015) is proposed in marine sediment studies, applying a washing in found a unexpected low rRNA:rDNA ratio when trying to alkaline phosphate buffers followed by centrifugation prior to establish a method to measure individual microbial activity standard DNA extraction (Alawi et al. 2014; Lever et al. and these ratios were due to high amounts of amplifiable 2015). During sampling of exDNA from water samples, a exDNA. Both results suggest that the exDNA fraction, which filtration through filters retaining the exDNA is required and is suitable in its quality for a qPCR or other downstream mo- it was found that the binding of exDNA is significantly dif- lecular methods, seems to derive to a large part from actively fering with filter material, pore size and several water quality released DNA and might thus reflect microbial activity, whilst parameters such as pH or total suspended solids (Liang and the exDNA deriving from lysed cells is not yielded using these Keeley 2013). methods. These results should be considered when applying 6350 Appl Microbiol Biotechnol (2018) 102:6343–6356 methods such as the viability PCR (Emerson et al. 2017; Novotny et al. 2016; Rocco et al. 2017) in order to damage Nocker et al. 2006; Wagner et al. 2008) or a treatment with structural integrity and consequently increase susceptibility of DNase I/proteinase K (Villarreal et al. 2013). These methods the biofilm constituents to antibiotic agents. Similarly, several are based on the assumption that exDNA mainly derives from genes associated with the release of exDNA or with autolysis dead cells. Consequently, iDNA and total DNA are measured as well as quorum sensing inhibitors can be the target of an by a degradation of the exDNA in one of two parallel samples anti-biofilm therapy (e.g. Bao et al. 2015; Beltrame et al. to give a live/dead ratio. In fact, exDNA may not only have 2015;Sietal. 2015; reviewed by Wolska et al. 2016). A derived from recently lysed and active cells but may also be nanomaterial cleaving exDNA of S. aureus biofilms was also relic DNA that has persisted outside of intact cell membranes proposed as a promising therapeutic material against biofilms for decades and centuries, especially when bound to inorganic (Thiyagarajan et al. 2016). particles such as soil colloids. Thus, an activity tracking using Deduced from its role as a main constituent of the EPS in exDNA should be further investigated considering this ancient biofilms, exDNA has been identified as a key contributor to exDNA probably being present at a low but stable rate in a uranium biomineralisation. It has been stated that the use of variety of environments. microorganisms producing exDNA in their biofilm may pro- vide a cheap alternative to standard physiochemical treatment exDNA as specific target matrix for (prokaryotic processes during the remediation of sites contaminated with and eukaryotic) biodiversity survey studies radionuclides (Hufton et al. 2016). In medical sciences, exDNA provides a useful tool for di- Within the field of environmental DNA research (Thomsen agnostics as well as therapy monitoring, as its concentrations and Willerslev 2015), a recent approach focused on the extra- correlate with a variety of pathologies including cancer cellular fraction of environmental DNA and aimed to study (Laktionov et al. 2004) and autoimmune disorders (Raptis the soil biodiversity at large scale (landscape scale; e.g. vege- and Menard 1980;reviewed by O’Driscoll 2007). Some stud- tation map) from large and thus representative sample vol- ies also highlight the possibility to use DNase I to treat tumour umes by applying a metabarcoding approach (e.g. Orwin et cells as it targets the exDNA that facilitates the aggregation of al. 2018; Taberlet et al. 2012b). However, quantitative as well the cells (Alekseeva et al. 2017; Hawes et al. 2015). During as qualitative conclusions should be interpreted with caution, pregnancy, the entire foetal genome circulates in the maternal as the results might be influenced by actively released and blood, enabling the non-invasive detection of foetal genetic ancient exDNA. disorders (Fan et al. 2012). Interestingly, exDNA has also been found to be useful in exDNA as tool for evolution research forensics: using chemical force microscopy, exDNA can be located and quantified on the surface of human epithelial cells In the field of marine biology, the identification and enumer- or on other surfaces, after a transfer through contact with skin ation of microscopic remains in sediments such as fossilised and saliva. In that way, it provides a new tool in the forensic protists can be supported studying ancient exDNA (aDNA), analysis of touch samples (Wang et al. 2017). being reported from sediments under anoxic, but also oxic Concluding, it can be stated that exDNA was often attrib- conditions and can date back to the Holocene and uted to mainly derive from dead cells; it has been shown that Pleistocene (Agnelli et al. 2007; Lejzerowicz et al. 2013). actively released exDNA makes up a quantitatively relevant Such data can be useful to give insights into the evolutionary fraction of the total exDNA pool of different environments. history of the studied species but have also been used to track An active release also goes hand in hand with a better pro- human activities along the shores of an alpine lake (Giguet- tection of the exDNA against DNases through the binding on Covex et al. 2014). different extracellular compartments such as minerals, lipids and proteins or through methylation (Böckelmann et al. exDNA as a target for biofilm treatment 2006). Once arranged to the desired structure, such extracel- lular exDNA-containing complexes can perform a number Representing an attractive target for biofilm control, exDNA of tasks in different environments, owed either to the sticky has been extensively studied and reviewed (e.g. Okshevsky character of the electrically charged exDNA molecule, or to and Meyer 2015; Okshevsky et al. 2015; Penesyan et al. 2015; the information that the exDNA can bear for other cells (Fig. Wnorowska et al. 2015). Next to its digestion with DNase 1). Next to these functions, exDNA can also serve as a (Aung et al. 2017; Bhongir et al. 2017; Brown et al. 2015a; source of energy and nutrients to other cells after a fragmen- Brown et al. 2015b; Rajendran et al. 2014; Waryah et al. 2017; tation by DNases. All these properties of exDNA provide a Ye et al. 2017), also the use of antibodies to target the DNA- great variety of possible applications that have been devel- binding proteins (DNABII) located at the vertex of crossed oped or are being developed across different fields of exDNA strands was proposed (Brockson et al. 2014; research. Appl Microbiol Biotechnol (2018) 102:6343–6356 6351 Acknowledgements We would like to thank Lara Insam for proof read- Biochem Function 2(1):33–37. https://doi.org/10.1002/cbf. ing and Dr. Alexander Steinbüchel, Editor-in-Chief of Applied 290020110 Microbiology and Biotechnology, for inviting us to write this review. Anker P, Mulcahy H, Qi Chen X, Stroun M (1999) Detection of circulat- ing tumour DNA in the blood (plasma/serum) of cancer patients. Cancer Metast Rev 18(1):65–73. https://doi.org/10.1023/a: Funding Information Open access funding provided by University of Innsbruck and Medical University of Innsbruck. Ascher J, Ceccherini MT, Guerri G, Nannipieri P, Pietramellara G (2009a) Be-MOTION^ of extracellular DNA (e-DNA) in soil. Fresenius Compliance with ethical standards Environ Bull 18(9A):1764–1767 Ascher J, Ceccherini MT, Pantani OL, Agnelli A, Borgogni F, Guerri G, Nannipieri P, Pietramellara G (2009b) Sequential extraction and Animal studies This article does not contain any studies with human genetic fingerprinting of a forest soil metagenome. Appl Soil Ecol and animal subjects. 42(2):176–181. https://doi.org/10.1016/j.apsoil.2009.03.005 Aucamp J, Bronkhorst AJ, Badenhorst CPS, Pretorius PJ (2016) A his- Conflict of interest The authors declare that they have no conflict of torical and evolutionary perspective on the biological significance of interest. circulating DNA and extracellular vesicles. 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