Arfaioli, Paola

Ascher, Judith

Borgogni, Federica

Ceccherini, Maria Teresa

Pietramellara, Giacomo

English

Florence Research

  EFFECT OF HEAVY METAL CATIONS ON THE FATE OF EXTRACELLULAR DNA ADSORBED AND BOUND ON CLAY MINERALSAscher J., Ceccherini M.T., Borgogni F., Arfaioli, P. and Pietramellara G.Department of Plant, Soil and Environmental Sciences, University of Florence , ItalyFusi et al.  (1989)  Soil Biol Biochem 21:911-920; Oedas (1984)  Clays and clay minerals 32:49-57; Pietramellara et al. (2007)  Biol Fertil Soils 43:731-739; Pietramellara et al. (2009) Biol Fertil Soils 45:219-235; Svarachorn et al. (1989) Appl Microbiol Biotechnol 30:299-304.The presence of high-valent metal cations on clay mineral surfaces is hypothesised to induce conformational changes in the secondary and tertiary structure of the DNA molecule adsorbed and bound onto clays, defined as M-conformation, and its condensation. The hypothesis that these reversible phenomena could enhance the resistance of DNA to enzymatic degradation strongly encourages the studies on the effects of heavy metal contamination in clay rich soils on the fate of extracellular soil DNA (eDNA). This lack of knowledge is relevant concerning the ecological role of soil eDNA in terms of persistence and availability for bacterial horizontal gene transfer by natural transformation, and as substrate for biofilm formation (Pietramellara et al., 2009). We assessed the effect of ionic Fe polymers on the adsorption (loosely versus tightly bound) of eDNA on clay minerals (dirty clay), that represent the mineral fraction of soil colloids. DNA-clay complexes (dirty eDNA/pure clay versus dirty eDNA/dirty clay) were analysed in terms of strength of DNA-clay interaction (adsorption isotherms). Challenging to conduct DNA-clay interaction studies in conditions as natural as possible, dirty  eDNA was extracted by simulating natural cell lysis without any purification, in order to avoid possible bias coming from DNA purification processes. DNA-clay interaction studies have been mostly performed under artificial conditions (pure DNA/pure clay). The performance under more natural conditions   (dirty  DNA/pure  clay) suggested a positive effect  of cellular debris coating DNA on DNA adsorption behaviors (Pietramellara et al., 2007). The present study, simulating natural soil conditions (dirty DNA/dirty clay), provided evidences of different amounts of DNA loosely (Fig. 1) and tightly bound (Fig. 2) on pure and dirty clay minerals, respectively. Lower amounts of loosely and higher amounts of tightly bound DNA on dirty clay with respect to pure clay have been observed (Fig. 3), suggesting Fe polymer-induced differences in the strength of interaction of DNA with clays. The proposed approach is capable to address a so far neglected aspect of heavy metal (HM) pollution, especially of clay-rich soils, namely the impact of HM cations coating soil colloid surfaces on the fate of eDNA in soil, providing relevant information on risk assessment of HM contaminated soils.  pure clay: Ca2+-Montmorillonite (M; Wayoming <2μm; Fusi et al., 1989). dirty clay: Ca2+-M coated with Fe(NO3)3 (Oades, 1984).  dirty  DNA was extracted from Bacillus subtilis  BD1512 by modifying the protocol of Svarachorn et al. (1989) and classified as dirty  DNA with cellular debris (dDNA+deb; Pietramellara et al., 2007).   Adsorption isotherms  were performed with 0.25 mg of a) pure  and b) dirty  clay, by adding 0.25, 0.5, 1.0, 2.5, 5.0 μg of dirty DNA (Pietramellara et al., 2007).The fractions of DNA 1) not adsorbed, 2) loosely bound and 3) tightly  bound  on clay minerals were determined based on fluorimetry (Qubit). The adsorption isotherm data were fitted to the Freundlich and Langmuir equations.Preliminary resultsµg  DNA tightly bound/clay 0,25         0,5            1             2,5            5Amounts of added dirty DNA (µg) / clay complexdirty DNA tightly bound on dirty claydirty DNA tightly bound on pure clay   0,25           0,5             1               2,5            5µg  DNA loosely bound/clayAmounts of added dirty DNA (µg) / clay complexdirty DNA loosely bound on dirty claydirty DNA loosely bound on pure clayFig. 3  Amounts of loosely and tightly bound dirty DNA on pure versus dirty clay.pure     dirtyclay Molecular weight of differently pure DNA fractions assessed by agarose gel electrophoresis. Dirty  DNA (dDNA)  coated with cellular debris (+deb) was used for interaction studies with pure  clay (Ca2+- M) and dirty  clay(Ca2+-M coated with ionic Fe). M1, M2 are standardized DNA markers.pure DNA        dDNA -deb     dDNA +deb      M1     M2      20 kb10 kbdirty DNA0510152025300 1 2 3 4 5 6Cs(µg –1 DNA mg –1 clay)0,25  0,5    1,0 2,5 5Ce (µg –1 DNA ml –1)dirty DNA – ADSORBED - dirty clayCs(µg –1 DNA mg –1 clay)Fig. 1  Adsorption isotherms of dirty  DNA and pure  versus dirty  clay showing different binding behaviors (loosely bound) as function of absence and presence of Fe polymers on clay mineral surfaces.0501001502002503003500 1 2 3 4 5 6Cs(µg –1 DNA mg –1 clay)dirty DNA - ADSORBED - pure clay0,25  0,5       1,0 2,5                 5Ce (µg –1 DNA ml –1 supernatant) Cs(µg –1 DNA mg –1 clay)Cs(µg –1 DNA mg –1 clay))Fig. 2  Binding behavior (tightly bound) of dirty DNA on pure versus dirty clay as function of absence and presence of Fe polymers on clay mineral surfaces.050010001500200025003000350040000 1 2 3 4 5 6Cs(µg –1 DNA mg –1 clay) dirty DNA - BOUND - pure clay0,25  0,5        1,0 2,5 5Ce (µg –1 DNA ml –1)Cs(µg –1 DNA mg –1 clay)Cs(µg –1 DNA mg –1 clay)01000200030004000500060000 1 2 3 4 5 6Cs(µg –1 DNA mg –1 clay) dirty DNA - BOUND - dirty clay0,25  0,5    1,0 2,5 5Ce (µg –1 DNA ml –1 supernatant)Ce (µg –1 DNA ml –1)Cs(µg –1 DNA mg –1 clay)0200030004000500060000 1 2 3 4 5 6Cs(µg –1 DNA mg –1 clay) dirty  -  - dirty clay0,25  0,5    1,0 2,5 5 (  –1  l –1 s r t t) (  –1  l –1)Cs(µg –1 DNA mg –1 clay)

Effect of heavy metal cations on the fate of extracellular DNA adsorbed and bound on clay minerals.

https://flore.unifi.it/bitstream/2158/852294/1/ICOBTE%202011%20Ascher%20et%20al.%202011.pdf

Effect of heavy metal cations on the fate of extracellular DNA adsorbed and bound on clay minerals.

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