EFFECT OF HEAVY METAL CATIONS ON THE FATE OF
EFFECT OF HEAVY METAL CATIONS ON THE FATE OF
EXTRACELLULAR DNA ADSORBED AND BOUND ON CLAY
EXTRACELLULAR DNA ADSORBED AND BOUND ON CLAY
MINERALS
MINERALS
Ascher J., Ceccherini M.T., Borgogni F., Arfaioli, P.
Ascher J., Ceccherini M.T., Borgogni F., Arfaioli, P.
and Pietramellara G.
and Pietramellara G.
Department of Plant, Soil and Environmental Sciences, University of Florence
Department of Plant, Soil and Environmental Sciences, University of Florence
, Italy
, Italy
Fusi et al.
Fusi et al. (1989)(1989) Soil Biol Biochem 21:911-920; Soil Biol Biochem 21:911-920; Oedas (1984)Oedas (1984) Clays and clay minerals 32:49-57; Clays and clay minerals 32:49-57; Pietramellara et al. (2007)Pietramellara et al. (2007) Biol Fertil Biol Fertil Soils 43:731-739; Soils 43:731-739;
Pietramellara et al. (2009)
Pietramellara et al. (2009) Biol Fertil Soils 45:219-235; Biol Fertil Soils 45:219-235; Svarachorn et al. (1989)Svarachorn et al. (1989) Appl Microbiol Biotechnol 30:299-304. 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 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 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 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 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 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). 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
We assessed the effect of ionic Fe polymers on the adsorption (loosely versusversus tightly bound) of eDNA on clay minerals ( tightly bound) of eDNA on clay minerals (dirtydirty clay), that clay), that represent the mineral fraction of soil colloids. DNA-clay complexes (
represent the mineral fraction of soil colloids. DNA-clay complexes (dirtydirty eDNA/pure clay versus eDNA/pure clay versus dirtydirty eDNA/ eDNA/dirtydirty clay) were analysed in clay) were analysed in terms of strength of DNA-clay interaction (adsorption isotherms). Challenging to conduct DNA-clay interaction studies in conditions as terms of strength of DNA-clay interaction (adsorption isotherms). Challenging to conduct DNA-clay interaction studies in conditions as
natural as possible,
natural as possible, dirtydirty eDNA was extracted by simulating natural cell lysis without any purification, in order to avoid possible bias eDNA was extracted by simulating natural cell lysis without any purification, in order to avoid possible bias coming from DNA purification processes.
coming from DNA purification processes.
DNA-clay interaction studies have been mostly performed under
DNA-clay interaction studies have been mostly performed under
artificial
artificial
conditions
conditions
(
(
pure
pure
DNA/
DNA/
pure
pure
clay). The performance
clay). The performance
under
under
more natural conditions
more natural conditions
(
(
dirty
dirty
DNA
DNA
/pure
/pure
clay) suggested a
clay) suggested a
positive effect
positive effect
of cellular debris coating DNA on DNA
of cellular debris coating DNA on DNA
adsorption behaviors (Pietramellara et al., 2007).
adsorption behaviors (Pietramellara et al., 2007).
The present study, simulating
The present study, simulating
natural soil conditions
natural soil conditions
(
(
dirty
dirty
DNA
DNA
/
/
dirty
dirty
clay
clay
), provided evidences of different amounts of DNA
), provided evidences of different amounts of DNA
loosely (Fig. 1) and tightly bound (Fig. 2) on
loosely (Fig. 1) and tightly bound (Fig. 2) on
pure
pure
and
and
dirty
dirty
clay minerals, respectively. Lower amounts of loosely and higher
clay minerals, respectively. Lower amounts of loosely and higher
amounts of tightly bound DNA on
amounts of tightly bound DNA on
dirty
dirty
clay with respect to
clay with respect to
pure
pure
clay have been observed (Fig. 3), suggesting Fe polymer-
clay have been observed (Fig. 3), suggesting Fe
polymer-induced differences in the strength of interaction of DNA with clays.
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
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
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.
information on risk assessment of HM contaminated soils.
purepure clay: Ca clay: 2+-Montmorillonite (M; Wayoming <2μm; Fusi et al., 1989).
dirtydirty clay: Ca clay: 2+-M coated with Fe(NO
3)3 (Oades, 1984).
dirtydirty DNA was extracted from Bacillus subtilis BD1512 by DNA
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) Adsorption isotherms
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 results Preliminary results µ g D N A ti gh tly bou nd/ cl ay 0,25 0,5 1 2,5 5 Amounts of added dirty DNA (µg) / clay complex
dirty DNA tightly bound on dirty clay dirty DNA tightly bound on pure clay
0,25 0,5 1 2,5 5 µ g D N A lo osel y bound/ cl ay
Amounts of added dirty DNA (µg) / clay complex
dirty DNA loosely bound on dirty clay dirty DNA loosely bound on pure clay
Fig. 3 Amounts of loosely and tightly bound
Fig. 3 Amounts of loosely and tightly bound
dirty DNA on
dirty DNA on purepure versus versus dirtydirty clay. clay.
pure
pure dirtydirty
clay
clay
Molecular weight of differently pure DNA fractions
assessed by agarose gel electrophoresis. DirtyDirty DNA DNA
(dDNA)
(dDNA) coated with cellular debris (+deb) was used for
interaction studies with pure clay (Ca2+- M) and dirtydirty clay clay
(Ca2+-M coated with ionic Fe). M1, M2 are standardized
DNA markers.
pure DNA dDNA -deb dDNA +debDNA +deb M1 M2
20 kb 10 kb
dirty
dirty DNA DNA
0 5 10 15 20 25 30 0 1 2 3 4 5 6 C s (µ g –1 D NA m g – 1 cl ay ) 0,25 0,5 1,0 2,5 5 Ce (µg –1 DNA ml –1)
dirty DNA – ADSORBED - dirty clay
0 5 10 15 20 25 30 0 1 2 3 4 5 6 C s (µ g –1 D NA m g – 1 cl ay ) 0,25 0,5 1,0 2,5 5 Ce (µg –1 DNA ml –1) 0,25 0,5 1,0 2,5 5 Ce (µg –1 DNA ml –1)
dirty DNA – ADSORBED - dirty clay
Fig. 1 Adsorption isotherms of
Fig. 1 Adsorption isotherms of dirtydirty DNA and DNA and purepure versus versus dirtydirty clay showing clay showing different binding behaviors (
different binding behaviors (loosely boundloosely bound) as function of absence and ) as function of absence and presence of Fe polymers on clay mineral surfaces.
presence of Fe polymers on clay mineral surfaces.
0 50 100 150 200 250 300 350 0 1 2 3 4 5 6 C s (µ g –1 D N A m g –1 cl ay
) dirty DNA - ADSORBED - pure clay
0,25 0,5 1,0 2,5 5 Ce (µg –1 DNA ml –1 supernatant) 0 50 100 150 200 250 300 350 0 1 2 3 4 5 6 C s (µ g –1 D N A m g –1 cl ay ) 0 50 100 150 200 250 300 350 0 1 2 3 4 5 6 C s (µ g –1 D N A m g –1 cl ay
) dirty DNA - ADSORBED - pure clay
0,25 0,5 1,0 2,5 5
Ce (µg –1 DNA ml –1 supernatant) )
Fig. 2 Binding behavior (
Fig. 2 Binding behavior (tightly boundtightly bound) of ) of dirtydirty DNA on DNA on purepure versus versus dirtydirty clay as clay as function of absence and presence of Fe polymers on clay mineral surfaces.
function of absence and presence of Fe polymers on clay mineral surfaces.
0 500 1000 1500 2000 2500 3000 3500 4000 0 1 2 3 4 5 6 C s (µ g –1 D N A m g – 1 cl a
y) dirty DNA - BOUND - pure clay
0,25 0,5 1,0 2,5 5 Ce (µg –1 DNA ml –1) 0 500 1000 1500 2000 2500 3000 3500 4000 0 1 2 3 4 5 6 C s (µ g –1 D N A m g – 1 cl a y) 0 500 1000 1500 2000 2500 3000 3500 4000 0 1 2 3 4 5 6 C s (µ g –1 D N A m g – 1 cl a
y) dirty DNA - BOUND - pure clay
0,25 0,5 1,0 2,5 5 Ce (µg –1 DNA ml –1) 0 1000 2000 3000 4000 5000 6000 0 1 2 3 4 5 6 C s (µ g –1 D N A m g – 1 cl ay
) dirty DNA - BOUND - dirty clay
0,25 0,5 1,0 2,5 5
Ce (µg –1 DNA ml –1 supernatant)
Ce (µg Ce (µg –1 DNA ml –1 DNA ml –1 supernatant)–1)
0 1000 2000 3000 4000 5000 6000 0 1 2 3 4 5 6 C s (µ g –1 D N A m g – 1 cl ay
) dirty DNA - BOUND - dirty clay
0,25 0,5 1,0 2,5 5
Ce (µg –1 DNA ml –1 supernatant)
Ce (µg Ce (µg –1 DNA ml –1 DNA ml –1 supernatant)–1)
0 1000 2000 3000 4000 5000 6000 0 1 2 3 4 5 6 C s (µ g –1 D N A m g – 1 cl ay
) dirty DNA - BOUND - dirty clay
0,25 0,5 1,0 2,5 5
Ce (µg –1 DNA ml –1 supernatant)
Ce (µg Ce (µg –1 DNA ml –1 DNA ml –1 supernatant)–1)
0 1000 2000 3000 4000 5000 6000 0 1 2 3 4 5 6 C s (µ g –1 D N A m g – 1 cl ay
) dirty DNA - BOUND - dirty clay
0,25 0,5 1,0 2,5 5
Ce (µg –1 DNA ml –1 supernatant)
