As eDNA is abundant in the local environment, micro-organisms have evolved to produce extracellular deoxyribonuclease enzymes. DNase enzymes catalyse the hydrolysis of the phosphodiester bond that links the phosphate of one nucleotide with the sugar 3’ carbon of the next nucleotide in the chain. The cleavage of nucleotides can either occur within the DNA strand (endonuclease) or at the ends of single stranded DNA (exonuclease).
In mammals, DNase I is an endonuclease that is universally produced by pancreatic cells. It has been heavily studied, and it understood to be involved in several processes, including, digesting DNA for nutrition (Lu et al., 2003), eDNA waste management (Samejima and Earnshaw, 2005), and clearing DNA during apoptosis (Oliveri et al., 2001). The activity of DNase I is dependent on the presence of both Ca2+ and Mg2+. In particular, Ca2+ binds tightly to DNase I and stabilises its active conformation. Without Ca2+, DNase I activity is negligible (Price, 1975). DNase I activity is highest at physiological pH (7.3-7.4), and is inhibited by sodium dodecyl sulphate (SDS), ethylene glycol tetraacetic acid (EGTA) (Price, 1975), actin (Lazarides and Lindberg, 1974), and many other substances. Importantly, the activity, function, and structure of microbial DNase enzymes is incredibly varied. As a result, it is not possible to infer a great deal from DNase enzymes, unless a novel one shares close homology to already characterised nuclease enzymes. However, DNase I because it has been extensively studied does provide a basis of DNase enzyme knowledge.
20 Many species of micro-organisms produce DNase enzymes that are released from the cell, or are bound to the cell-wall-surface. As extracellular DNase production is genus or species specific, it has long been used as a way to diagnostically determine a cultured isolate. For instance, DNase testing (e.g. DNase test agar) is often used to distinguish S.
aureus from coagulase-negative staphylococci. Furthermore, in the 1950s Weckman
and Catlin (1957) suggested that DNase tests could be a useful characteristic to study bacterial taxonomy. These authors also found that the supernatant fluid of S. aureus cultures contained highest DNase activity, suggesting that they were studying non-cell- wall-bound nuclease enzymes. This interesting finding occurred because the DNase test, which determined the viscosity of DNA after incubation with culture fluids, allowed the use of broth cultures, supernatants, and cell pellets. There are many different tests for DNase activity, with the common procedure being the growth of micro-organisms on DNase test agar. This test is quick, but cannot provide detailed information such as enzyme location in relation to the cell. The FRET nuclease activity assay developed by Kiedrowski et al., (2011) to study S. aureus Nuc1 is another approach to studying extracellular DNases. This assay uses a short qPCR probe with a fluorophore attached to the 5’ end, and a fluorescence quencher on the 3’ end. Once the probe is cleaved by a DNase enzyme, quenching of fluorescence ceases and light emission can be quantified with a microplate reader. Other DNase tests include in gel zymography, and agarose gel electrophoresis after DNA digestion (Shak et al., 1990).
Extracellular DNase enzymes are produced by a diverse range of micro-organisms. Fungi, such as Cryptococcus neoformans, can produce extracellular nucleases, although this trait is genus specific (Cazin et al., 1969). A wide-range of periodontal bacteria have been tested, and 27 out of 34 produced DNase activity (Palmer et al., 2012). Clinical isolates of anaerobic micro-organisms, including Fusobacterium spp., very commonly produce extracellular DNase enzymes (Porschen and Sonntag, 1974). Again, extracellular nuclease activity was often genus specific amongst anaerobic micro- organisms. Many species of streptococci also possess extracellular DNases, including S.
pneumoniae (Hasegawa et al., 2010), Streptococcus pyogenes (Zhu et al., 2013), and Streptococcus suis (Fontaine et al., 2004). Extracellular DNase production also appears
to be a key characteristic of S. aureus (Tang et al., 2011) and P. aeruginosa (Mulcahy et
al., 2010). Ultimately, it appears as though this trait of micro-organisms is very
common.
Extracellular DNA is a ubiquitous macromolecule, and its ubiquity is the likely reason for the sheer diversity of micro-organisms that produce one, or more DNase
21 Table 1.1 Extracellular bacterial nucleases and their proposed functions.
Micro-organism Protein Size (aaa) Conserved Domains Proposed Function References
Streptococcus gordonii SsnA 779 MnuA domain, OBF (1) Unknown This thesis
Streptococcus suis SsnA 1041 EEP domain, OBF (3) Virulence (Fontaine et al., 2004)
Streptococcus pyogenes Spd1 252 Non-specific nuclease
domain Virulence (Korczynska et al., 2012)
SpnA 910 EEP domain, OBF (3) Virulence (Hasegawa et al., 2010;
Chang et al., 2011)
Sda1 390 Non-specific nuclease
domain NET evasion (Buchanan et al., 2006)
Streptococcus
pneumoniae EndA 248
Non-specific nuclease
domain NET evasion
(Midon et al., 2011; Zhu et
al., 2013) Streptococcus
agalactiae NucA 261
Non-specific nuclease
domain NET evasion (Derré-Bobillot et al., 2013)
Staphylococcus aureus Nuc1 215 SNc domain NET Evasion and biofilm
formation
(Berends et al., 2010; Kiedrowski et al., 2011;
Beenken et al., 2012)
Nuc2 177 SNc domain Biofilm formation and
virulence
(Beenken et al., 2012; Kiedrowski et al., 2014)
22
Vibrio cholerae Dns 231 EndA domain NET evasion, transformation
and biofilm formation
(Blokesch and Schoolnik, 2008; Seper et al., 2011;
Seper et al., 2013)
Xds 869 EEP domain, OBF (1) NET evasion and biofilm
formation
(Seper et al., 2011; Seper et
al., 2013) Shewanella oneidensis ExeM 871 EEP domain, OBF (1) Biofilm formation (Heun et al., 2012)
ExeS 948 MnuA domain, OBF (1) Biofilm formation (Heun et al., 2012)
EndA 258 EndA domain eDNA digestion (Heun et al., 2012)
Pseudomonas
aeruginosa EddB 779 MnuA domain, OBF (1) eDNA digestion (Mulcahy et al., 2010)
Neisseria gonorrhoeae Nuc 233 SNc domain Biofilm formation (Steichen et al., 2011)
Mycoplasma genitalium MG186 250 SNc domain eDNA digestion (Li et al., 2010)
a
Amino acid residues
MnuA, membrane-associated nuclease of Mycoplasma pulmonis homologue; OBF, oligonucleotide/oligosaccharide fold homologue; EEP,
exonuclease-endonuclease-phosphatase catalytic domain homologue; NucA/NucB, deoxyribonuclease NucA/NucB homologue; EndA, endonuclease I homologue; SNc, staphylococcal nuclease homologue;
23 enzymes. There are many proposed functions for these enzymes, and it is possible that each DNase could have a number of roles. A selection of microbial DNases with their proposed functions is listed in Table 1.1. Many of the extracellular nucleases that are produced by pathogenic bacteria have been linked to virulence. A key factor that increases the virulence of DNase producing micro-organisms is the ability to degrade neutrophil extracellular traps (NETs). These networks of DNA and some globular protein domains are produced by neutrophils, and entrap bacteria (Brinkmann et al., 2004). It is therefore highly desirable for a pathogenic micro-organism to be able destroy NETs, thereby reducing their antimicrobial potential. This trait has been studied in a number of micro-organisms. When nuc-deficient strains of S. aureus USA 300 LAC were cultured in the presence of NETs they were significantly more likely to be entrapped by the DNA matrix than wild-type S. aureus (Berends et al., 2010).
Streptococcus agalactiae NucA can degrade NETs, but substitution of histidine148 by alanine impaired this function as it abolishes enzyme activity by altering the enzyme active site (Derré-Bobillot et al., 2013). Cell death caused by the host immune system during disease also releases large amounts of DNA into the extracellular milieu. Extracellular DNA can be found in the lungs of cystic fibrosis patients (Shak et al., 1990). It is unknown if DNase production helps disseminate micro-organisms through viscous mucus that contains DNA but biofilm dispersal has been suggested as a mechanism to promote pathogen transmission (Hall-Stoodley and Stoodley, 2005).
Interestingly, Shewanella oneidensis produces three extracellular nucleases, ExeM, ExeS, and EndA, with each having a distinct function. Deletion of EndA removed the ability of S. oneidensis to use eDNA as a source of phosphorus (Heun et al., 2012). ExeS and ExeM are involved in biofilm formation, as deletion of either gene affects biofilm formation (Gödeke et al., 2011). When stained with DDAO, eDNA can be visualized surrounding dense aggregations of cells in the exeM-deficient S. oneidensis biofilm. Other species of bacteria that can produce two or more DNase enzymes that are proposed to have differing roles include S. pyogenes (Sumby et al., 2005; Buchanan et
al., 2006; Hasegawa et al., 2010; Chang et al., 2011; Korczynska et al., 2012), and Vibrio cholerae (Seper et al., 2011; Seper et al., 2013). In V. cholerae the extracellular
nuclease, Dns, is involved with transformation (Blokesch and Schoolnik, 2008). As cell densities and the quorum-sensing regulator HapR increase Dns is repressed, leading to higher transformation frequencies. However, the other V. cholerae extracellular DNase, Xds has been linked to biofilm modulation through the degradation of DNA (Seper et
24
al., 2011). More recently, the same authors have proposed that Xds and Dns are
involved in facilitating NET escape (Seper et al., 2013).
Extracellular DNA may be an important bacterial nutrient in nutrient poor environments. Although DNA is an unstable molecule (Lindahl, 1993), it can persist for long periods of time. Recently, mitochondrial DNA was sequenced from a femur belonging to a Denisovan hominin that lived 400,000 years ago (Meyer et al., 2013). Therefore, eDNA is a nutrient source that bacteria can rely on, and DNase production can allow utilization of this substrate. Deoxyribonucleic acid consumption by bacteria was first observed in E. coli (Finkel and Kolter, 2001) and has since been shown in
Shewanella spp. (Pinchuk et al., 2008; Heun et al., 2012) and Helicobacter pylori
(Liechti and Goldberg, 2013). The Archaean species, Haloferax volcanii, may use intracellular DNA as a phosphorus storage polymer, and it is hypothesised by Zerulla et
al., (2014) that DNA may have evolved for this function. It is unknown whether micro-
organisms ingest biofilm matrix eDNA during periods of starvation.
If extracellular DNases are required for nutrient metabolism then they may be influenced by transcriptional regulators, in relation to available carbon sources. The major regulator of carbon source utilisation in streptococci, such as S. gordonii, is the carbon catabolite regulator, carbon catabolite protein A (CcpA) (Dong et al., 2004). This protein down-regulates genes encoding systems for utilization of less favourable carbon sources when more energetically favourable sources are available. There is evidence that CcpA also has a role in regulation of extracellular DNases. For instance, the gene of the cell-bound DNase of S. suis, SsnA, contains a catabolite responsive element (CRE) that is bound by CcpA, repressing ssnA during the stationary phase of cell growth (Willenborg et al., 2014). In S. pyogenes MGAS5005, the extracellular DNase, Spd, is regulated by CcpA (Shelburne et al., 2008). Gene transcription of a
ccpA-deficient strain of S. pyogenes was compared with strain MGAS5005 by
quantitative real time PCR. In times of nutrient limitation spd transcription was higher in the wild-type strain than the ccpA mutant, indicating that in this model CcpA is an activator of Spd in glucose-limited media, such as saliva. CcpA appears to regulate extracellular DNases in different environmental conditions, which may influence a number of DNase functions, including: (i) transformation, (ii) nutrient scavenging, (iii) virulence (e.g. NET degradation), (iv) host biofilm control, (v) competitor biofilm dispersal, and (vi) protection against the deleterious effects of eDNA. It is not yet known whether CcpA regulates extracellular DNases in oral streptococci.
25 The role of extracellular DNases during transformation and nutrient acquisition may be linked, and it has been suggested that competence evolved to meet the nutritional demands of bacteria. Extracellular dsDNA binds to cell-wall proteins, such as ComB in
B. subtilis (DNA uptake reviewed in detail by Dubnau, 1999). Internalization of DNA
relies on it being modified from dsDNA to ssDNA through the activity of a specific nuclease, such as EndA in S. pneumoniae (Lacks and Neuberger, 1975). DNA transporter proteins in the cell-wall facilitate ssDNA uptake, with the energy required coming from ATP. This process is therefore reliant on specific, cell-bound nucleases, such as EndA to convert dsDNA to ssDNA. It is possible that the strand released by nuclease activity could remain in the periplasm in Gram-negative bacteria, to be utilized as a nutrient source. However, it is likely that half the DNA is lost to the surrounding medium and it therefore represents a wasteful pathway for obtaining nutrition from DNA and more likely has the singular function of gene uptake. Non-specific nucleases, including NucB in B. subtilis, which are released into the extracellular milieu may be more plausible candidates for DNase enzymes involved in DNA metabolism. In this manner, non-specific nucleases may degrade dsDNA into a size that is able to permeate the cell wall, or allow further enzyme activity, such as extracellular phosphatases that can remove phosphorus groups from DNA. This is a phenomenon that occurs in
Shewanella spp. (Pinchuk et al., 2008). The role of extracellular DNases in DNA uptake
is likely to change depending on environmental pressures. Recently, S. pneumoniae EndA was shown to be secreted, and have anti-NET activity via a pathway that is independent of competence development (Zhu et al., 2013). Extracellular DNA uptake is complex, and it would be interesting to study the impact that strain, species and environmental variability has on the DNA uptake machinery used.
Given the clear role of eDNA in promoting biofilm adhesion and maintaining mature biofilm stability there have been a number of studies examining the role of extracellular DNases in releasing cells from biofilms. As already discussed S. oneidensis produces three extracellular nucleases that alter biofilm formation (Gödeke et al., 2011). Furthermore, biofilm extent is increased in S. aureus when nuc1 and nuc2 extracellular nuclease genes are deleted (Kiedrowski et al., 2011; Beenken et al., 2012). However, these in vitro experiments were not reproduced in a murine catheter model, where biofilm formation was decreased in nuc1 and nuc2 mutants compared to wild-type S.
aureus (Beenken et al., 2012). In addition, it has been proposed that extracellular
nuclease release may be responsible for biofilm dissemination in Bacillus licheniformis (Nijland et al., 2010). Recently, a nucB-deficient mutant of B. licheniformis was found
26 to produce a thicker biofilm during sporulation (Edward Mason, unpublished data). It was Akrigg and Mandelstam (1978) who first demonstrated that B. subtilis produces a DNase, NucB, during sporulation. It is thought that B. subtilis NucB degrades DNA released by mother cell lysis, at the latter stages of sporulation (Hosoya et al., 2007), and this may share homology with B. licheniformis NucB. Although the effects of extracellular DNases against the host micro-organism have been studied it is so far unknown if these enzymes are used to compete against other micro-organisms in mixed- species biofilms. Many micro-organisms rely on eDNA for promoting biofilm formation, and it therefore seems plausible that bacteria such as S. aureus could reduce the colonisation of eDNA-producing bacteria.
Extracellular DNases may also protect against the deleterious effects of eDNA on microbes. For instance, eDNA can bind and sequester divalent metal cations, like Mg2+ and Ca2+. Removal of these cations from the bacterial cell surface, by eDNA, can lead to cell lysis (Mulcahy et al., 2010). Furthermore, bacterial DNA can alert the host immune system to the presence of foreign micro-organisms (Hemmi et al., 2000). Therefore, invading bacteria could mask themselves by degrading their own eDNA. Lastly, eDNA inhibits biofilm development in Salmonella enterica (Wang et al., 2013). Biofilm formation was restored when abiotic surfaces were treated with DNase I.
In conclusion, persistence of DNA in the environment has led to many possible functions for extracellular DNase enzymes. The most important roles of these enzymes are likely facilitating NET degradation, consumption of DNA as a nutrient source, and regulating biofilm formation. A neglected aspect of research is biofilm competition. For instance, many periodontal bacteria produce DNase enzymes (Palmer et al., 2012), and this could re-shape the mixed-species biofilms that form in the oral cavity.