1 2 DNA FRAGMENTATION IN MICROORGANISMS...

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DNA FRAGMENTATION IN MICROORGANISMS ASSESSED IN SITU 2

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Running title: DNA fragmentation in microorganisms 5

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José Luis Fernández a, b *

, Mónica Cartelle a

, Lourdes Muriel a

, Rebeca Santiso a, b

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María Tamayo a, b

, Vicente Goyanes a

, Jaime Gosálvez c, Germán Bou

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a INIBIC-Genética, Complejo Hospitalario Universitario Juan Canalejo, As Xubias 84, 11

15006- A Coruña, Spain. 12

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Laboratorio de Genética Molecular y Radiobiología, Centro Oncológico de Galicia, 14

Avda. de Montserrat s/n, 15009-A Coruña, Spain. 15

16 c Unidad de Genética, Facultad de Biología, Universidad Autónoma de Madrid, 28049-17

Madrid, Spain. 18

19 d INIBIC-Microbiología, Complejo Hospitalario Universitario Juan Canalejo, As 20

Xubias, 84, 15006-A Coruña, Spain 21

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27 * Correspondence 28

Dr. José Luis Fernández 29

Sección de Genética y Unidad de Investigación, 30

Complejo Hospitalario Universitario Juan Canalejo, 31

As Xubias, 84, 32

15006- A Coruña, Spain 33

Tel: 34 981 287499 34

Fax: 34 981 287122 35

E-mail: [email protected] ; [email protected] 36

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00318-08 AEM Accepts, published online ahead of print on 8 August 2008

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ABSTRACT 37

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Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. 39

Unlike research in higher eukaryotic cells, DNA fragmentation in microorganisms is 40

rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, 41

which allows for simple and rapid discrimination of bacteria with fragmented DNA. 42

Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer 43

to partially remove the cell walls, membranes, and proteins, and then stained with a 44

DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses 45

peripheral diffusion of DNA fragments. Cells without DNA fragmentation only show 46

limited spreading of DNA fiber loops. These results have been seen in several Gram-47

negative and Gram-positive bacteria, as well as in yeast. Detection of DNA 48

fragmentation was confirmed by fluoroquinolone treatment and by DNA Breakage 49

Detection-Fluorescence In situ Hybridization (DBD-FISH). Proteus mirabilis with 50

DNA spontaneously fragmented during exponentially and stationary growth, or 51

Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics 52

such us ciprofloxacin or ampicillin, were clearly detected. Similarly, fragmented DNA 53

was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay 54

may be useful for a simple and rapid evaluation of DNA damage and repair as well as 55

cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial 56

agents, or environmental conditions. 57

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Key words: DNA fragmentation; DNA damage; cell death; quinolones; DBD-FISH59

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INTRODUCTION 60

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Chromosomal DNA fragmentation, resulting from massive DNA double-strand breaks, 62

is a hallmark of cell death. In higher eukaryotic cells, this may be a consequence of 63

active programmed cell death (PCD), i. e. apoptosis, where DNA is cleaved by an 64

activated endonuclease (26). Otherwise, DNA fragmentation may occur passively 65

through necrotic cell death. Passive DNA fragmentation is likely in microorganisms 66

killed by various causes. Recent studies suggest, however, a possible PCD in unicellular 67

bacteria and yeast (38). In fact, bactericidal antibiotics may trigger a PCD response. It 68

has been reported that bactericidal antibiotics can stimulate the production of hydroxyl 69

radicals that contribute to cell death (20). Tolerant bacterial cells, that are resistant to the 70

bactericidal action of some antibiotics, may be cells with a disabled PCD. Ecological 71

pressure, differentiation processes, starvation and certain DNA damaging agents, may 72

activate PCD involving DNA fragmentation (1, 6, 18, 22, 35). DNA fragments released 73

during autolysis may be absorbed by other bacteria, contributing to antigenic variation 74

and the spread of antibiotic resistance (12). Bacterial autolytic mechanisms have been 75

described primarily with reference to the cell wall level. Nevertheless, active DNA 76

fragmentation has only partially been addressed. 77

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The presence of DNA breakage is usually evaluated using biochemical or molecular 79

procedures such us alkaline unwinding, DNA elution, gel electrophoresis, sucrose 80

gradient sedimentation, melting curve analysis, viscoelastometry, or light-scattering (2, 81

37). Unfortunately, evaluating DNA fragmentation within an individual cell is not 82

possible nor is it possible to identify important low level intercellular variations. In situ 83

procedures allow for cells with fragmented DNA to be discriminated from those without 84

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DNA fragmentation. However, in situ procedures have been developed primarily for use 85

in higher eukaryotic cells. DNA breaks can be enzymatically labelled by attaching 86

modified nucleotides that can be visualized by direct and indirect methods (10). Usually 87

this process involves the E. coli DNA polymerase I in the In Situ Nick Translation 88

(ISNT) assay or end-labelling with the Klenow fragment of DNA polymerase I, or the 89

terminal deoxynucleotidyl transferase (TdT) in the TUNEL procedure (10). In each of 90

these, a free and accessible 3’-OH group at the end of the break is necessary as substrate 91

for extension, but the ends of DNA breaks may be chemically modified by many DNA-92

damaging agents. 93

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In situ detection of DNA breaks in higher eukaryotes can also be seen through the 95

single-cell gel electrophoresis (SCGE) or comet assay (27). In this technique, the cells 96

are trapped in an inert agarose microgel on microscope slide, deproteinized by 97

incubation with a lysing solution, and then electrophoresed. DNA staining with a 98

fluorochrome reveals a comet, with a head and a tail of chromatin in the direction of the 99

positive pole of the electric field. Cells with DNA breaks have a higher tail and/or 100

greater DNA concentration in the tail. In contrast to enzymatic labelling, cells in the 101

SCGE assay are unfixed, fully accessible to lysis, and the DNA migration is not 102

dependent on the chemical nature of the breaks. An image analysis system is habitually 103

used for evaluation. Nevertheless, electrophoresis is not necessary to identify cells with 104

fragmented DNA. After lysis in the agarose microgel, cells with fragmented DNA can 105

be identified because they produce a peripheral halo of diffused DNA fragments in the 106

agarose matrix. This is the underlying principle of the diffusion assay to detect cells 107

with DNA fragmentation (33, 36). 108

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Regarding microorganisms, the TUNEL assay has been used to determine DNA 110

fragmentation in spheroplasts from yeast, but only one paper has described its use in 111

bacteria, specifically in E. coli and in the archaeon Haloferax volcanii (31). The 112

procedure was time-consuming, requiring fixation, centrifugation and permeabilization 113

steps. Moreover, the evaluation used a flow cytometer, requiring a high number of cells. 114

These technical factors make this procedure impractical for routine assessment of DNA 115

fragmentation in the standard microbiology laboratory. For the comet assay, only one 116

paper has described its use for E. coli (34). The procedure was also lengthy, requiring 117

lysozyme digestion of the cell wall prior to incubation in the lysis solution and in 118

conjunction with prolonged proteinase K digestion. The resulting images are difficult to 119

interpret. 120

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Despite its importance from both a basic research and a clinical point of view, DNA 122

fragmentation has not been assessed in the microbiology laboratory. This may be due to 123

the lack of a simple and rapid evaluation procedure. Most current techniques are 124

complex, technically demanding, and are not specifically adapted for the different 125

microorganisms. Here we present a diffusion-based assay to identify DNA 126

fragmentation in bacteria and yeast, using fluorescence microscopy. This assay is 127

assembled as a kit in order to implement a simple, fast, reproducible and accurate 128

method for studying DNA fragmentation in microorganisms. 129

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MATERIALS AND METHODS 130

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Microorganisms and cultures. Chromosomal DNA fragmentation was assayed in 132

Gram-negative and Gram-positive bacteria (Table 1). Gram-negative bacteria were 133

grown in Luria Bertani (LB) broth (1% Bacto Tryptone, 0.5% yeast extract, 0.5% NaCl) 134

or on LB agar, at 37ºC in aerobic conditions. Gram-positive bacteria were grown on 135

Trypticase Soy Agar (TSA) plates (Diagnostics Systems, Sparks, MD, USA). Candida 136

albicans and S. cerevisiae yeast were grown in yeast extract/peptone/dextrose (YPD) 137

broth and plates. Cell growth in liquid cultures was monitored with the 138

spectrophotometer (Unicam 8625, Cambridge, UK). In amphotericin B experiments, 139

viability was determined by colony-counting after sequential dilutions and plating. 140

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Experiments. Five different experiments illustrated the use of the procedure to 142

determine chromosomal DNA fragmentation. In the first experiment, aliquots of 143

exponentially growing cultures of the E. coli strain TG1, were exposed to ciprofloxacin 144

(5 µg/ml and 0.012 µg/ml), for 40 min in LB broth, at 37ºC. Fluoroquinolone is an 145

inducer of DNA fragmentation; this experiment validated the assay. In the second 146

experiment, P. mirabilis was incubated in LB medium at 37ºC in aerobic conditions. 147

The initial optical density in the spectrophotometer, measured at 600 nm, was 0.05. P. 148

mirabilis in exponentially growing phase and in stationary phase were analyzed for 149

frequency of cells with fragmented DNA and for membrane permeability, in aliquots 150

that were removed periodically along 106 h. These were batch cultures, where growth 151

uses an unsupplemented amount of nutrients, so the nutrients will decrease in time, 152

together with an increase in metabolites. In a third experiment, TG1, either 153

exponentially growing or in stationary phase, was incubated with 10mM hydrogen 154

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peroxide for 10 min at room temperature in LB broth. Immediately the cells were 155

processed with the DNA fragmentation kit, as described later. The purpose of this 156

experiment was two-fold: 1) to assess the DNA damage induced by hydroxyl radicals, 157

and 2) to explore the influence of the growth phase on the effect of hydrogen peroxide 158

on DNA. In the fourth experiment, exponentially growing cultures of TG1 were 159

exposed to ampicillin (300 µg/ml) for 40 min in LB broth, at 37ºC. This compared the 160

effect on DNA of incubation with an antibiotic with a different mechanism of action to 161

that of quinolones. Finally, exponentially growing cultures of S. cerevisiae were 162

incubated with increasing doses of amphotericin B (0, 0.5, 1, 2, 4, 8 and 16 µg/ml), at 163

30ºC, for 3 and 24 h, in suspension, in YPD broth. After incubation, the cells were 164

processed with the DNA fragmentation kit, as described later. 1000-5000 165

microorganisms were scored per treatment. 166

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Membrane permeability. A mixture of SYBR Green II (21) and propidium iodide 168

(Molecular Probes, Eugene, OR, USA) was prepared in PBS, at 333X and 0.17 µg/ml, 169

respectively. A 4µl aliquot of microorganisms growing in liquid medium was diluted in 170

16 µl of culture medium and was incubated for 5 min with 4 µl of dye mixture in the 171

dark. If the microorganisms were at a very low density, the dye mixture was added to a 172

20 µl aliquot of cell culture without dilution. 5 µl of the stained cell suspension were 173

dropped onto a glass slide, covered with a coverslip, and examined by fluorescence 174

microscopy. Permeable cells that did not exclude the propidium iodide appeared red, 175

whereas those “alive” only fluoresced green. In S. cerevisiae, non-permeable cells 176

appeared unstained. 177

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Determination of DNA fragmentation. The commercial sperm-Halomax® kits 179

(Halotech DNA SL, Madrid, Spain) used to determine DNA fragmentation in different 180

mammalian spermatozoa were evaluated (13, 17, 30). Using sperm-Halomax® as a 181

reference, a prototype Micro-Halomax® kit was developed for microorganisms 182

(Halotech DNA SL, Madrid, Spain). While Gram-negative bacteria could be directly 183

processed, a previous brief cell wall digestion step was necessary for Gram-positive 184

bacteria. S. agalactiae was incubated with mutanolysin (0.1 mg/ml), E. faecalis with a 185

mix of mutanolysin (0.1 mg/ml) and lysozyme (4 mg/ml), and S. aureus with a mix of 186

lysostaphin (0.05 mg/ml) and lysozyme (0.25 mg/ml). S. pyogenes was incubated with 187

lysozyme (1 mg/ml). To this purpose, bacteria were scrapped from the culture plate and 188

resuspended in 0.25 ml of LB medium or phosphate buffer saline (PBS) pH 6.88, in 0.5 189

ml snap cap micro-centrifuge tubes. The enzyme was added at the desired final 190

concentration and was incubated for 15 min at 37ºC. C. albicans and S. cerevisiae were 191

digested with lyticase, 2.5 U/ml for 15 min and 0.07 U/ml for 10 min, respectively, in 192

1M sorbitol, 1M EDTA and 15mM betamercaptoethanol, pH 7.5, at 30ºC. All enzymes 193

were purchased from Sigma (St Louis, MN, USA). 194

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An aliquot of each sample was diluted to a concentration of 5-10x106 196

microorganisms/ml in the broth culture medium specific for each microorganism. The 197

yeast was centrifuged and resuspended in the lyticase buffer without the enzyme. 0.5 ml 198

snap cap micro-centrifuge tubes containing gelled aliquots of 60 µl of low-melting-point 199

agarose (Pronadisa, Laboratorios Conda, Madrid, Spain) in distilled water are provided 200

with the Micro-Halomax® kit. The tube was placed in a water bath at 90-100ºC for 5 201

min to melt the agarose and then transferred to a water bath at 37ºC (Memmert, 202

Schwabach, Germany). After 5 min incubation, to allow for equilibration to 37ºC, 25 µl 203

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of the diluted sample containing the microorganism was added to the tube and was 204

mixed with the melted agarose. Aliquots (20 µl) of the sample-agarose mixture were 205

pipetted onto a precoated slide provided with the kit and were covered with a 22 x 22 206

mm coverslip. The coating of the slides consists of a dried agarose layer prepared with 207

1% standard agarose in water and drying in an oven at 80ºC (Memmert, Schwabach, 208

Germany). The slide was placed on a cold plate in the refrigerator (4ºC) for 5 min, to 209

allow the agarose to solidify producing a microgel with the intact cells trapped inside. 210

The coverslip was gently removed, and the slide was immersed in 10 ml of lysis 211

solution provided in the Micro-Halomax® kit, previously tempered to 37 ºC, for 5 min, 212

in a closed try at 37ºC. This solution contains 2% SDS, 0.05M EDTA and 0.1M DTT, 213

pH 11.5. The slide was always placed in horizontal position to prevent DNA stretching. 214

After washing horizontally for 3 min in a tray with abundant distilled water, the slide 215

was dehydrated by incubation horizontally in cold (-20ºC) ethanol baths, first 70%, then 216

90% and finally 100%, for 3 min each, followed by air-drying in an oven (Memmert, 217

Schwabach, Germany). 218

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DNA staining with the fluorochrome SYBR Gold (Molecular Probes, Eugene, OR, 220

USA) (7), could be performed immediately after drying. Before staining, the dried slide 221

must be incubated in a microwave oven (Whirlpool, Norrköping, Sweden) at 750 W for 222

4 min to promote the attachment of DNA to the slide. The slide may also be placed in 223

an oven at 80ºC for 1h to overnight. The slide was then stained with 25 µl of SYBR 224

Gold diluted 1:100 in TBE buffer (0.09M Tris-Borate, 0.002M EDTA, pH 7.5), covered 225

with a plastic coverslip, and incubated for 5 min in the dark. The slide was briefly 226

washed and mounted in TBE. Fluorescence microscopy must be performed immediately 227

after staining to avoid drying. If needed, the slide may be stored at 4ºC for hours in a 228

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self-made humid box in the dark to prevent drying. If dried, the coverslip may be 229

removed by incubation in PBS and, after a brief wash, may be re-stained again. If 230

immediate evaluation is not necessary, dried slides may be left overnight or a couple of 231

days in a high temperature oven (80ºC) and then stored in a tightly closed box, in the 232

dark, at room temperature, for several months, before staining. 233

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Fluorescence microscopy allows for 10x to 100x magnification, but 100X is necessary 235

for a precise visualization of the small spots from nucleoids with fragmented DNA. 236

Three microgels can be placed on a same slide, including a control sample if required, 237

so that all microgels are simultaneously processed under the same conditions. 238

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DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) (14, 240

15). To confirm the presence of DNA breakage in cells with diffused DNA spots, the 241

DBD-FISH procedure was used in E. coli, P. mirabilis, Acinetobacter haemolyticus, 242

Staphylococcus aureus and S. cerevisiae nucleoids. The cells were immersed in agarose 243

microgels and were lysed as described. They were then washed in 0.9%NaCl and were 244

incubated in an alkaline unwinding solution (0.03M NaOH) for 2.5 min at 22ºC. The 245

gels were neutralized in 0.4M TrisHCl, pH 7.5, washed in distilled water, dehydrated in 246

increasing ethanol baths (70-90-100%) for 2 min each, and air-dried. 247

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A DNA probe to label the total DNA from the microorganism was prepared. DNA from 249

each microorganism was isolated using standard procedures, and was labeled with 250

biotin-16-dUTP, using a nick translation kit, according to the manufacturer’s 251

instructions (Roche Applied Science, San Cugat del Vallés, Spain). The DNA probe 252

was mixed at 4.3 ng/µl in the hybridization buffer (50% formamide / 2xSSC, 10% 253

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dextran sulfate, 100mM calcium phosphate, pH 7.0) (1xSSC is 0.015M NaCitrate, 254

0.15M NaCl, pH 7.0). The probe in hybridization buffer was denatured by incubation at 255

80ºC for 8 min and was then incubated on ice. This solution (30 µl) was pipetted onto 256

the dried slide, covered with a glass coverslip (22x60mm) and incubated overnight at 257

room temperature, in the dark, in a humid chamber. The coverslip was removed, and the 258

slides were washed twice in 50% formamide/2xSSC, pH 7.0, for 5 min, and twice in 259

2xSSC pH 7.0, for 3 min, at room temperature. The slides were incubated with 80 µl of 260

blocking solution (4XSSC, 0.1% Triton X-100, 5%BSA) for 5 min, covered with a 261

plastic coverslip, in a humid chamber, at 37ºC. This solution was decanted, and the 262

bound probe was detected by incubation with 80 µl of streptavidin-Cy3 (Sigma Chem, 263

St Louis, MN, USA) in 4XSSC, 0.1% Triton X-100, 1%BSA (1:200), covered with a 264

plastic coverslip, in a humid chamber at 37ºC. After washing in 4XSSC, 0.1% Triton X-265

100, three times, 2 min each, slides were counterstained with 20 µl of DAPI (2 µg/ml) 266

in Vectashield (Vector, Burlingame, CA). 267

Fluorescence Microscopy and Digital Image Analysis. Images were viewed with an

epifluorescence microscope (Nikon E800), with a 100x objective and appropriate

fluorescence filters for FITC-SYBR Gold (excitation 465-495 nm, emission 515-555

nm), PI-Cy3 (excitation 540/25 nm, emission 605/55 nm) and DAPI (excitation 340-380

nm, emission 435-485 nm). The images were captured with a high-sensitivity CCD

camera (KX32ME, Apogee Instruments, Roseville, CA). Groups of 16 bit digital

images were obtained and stored as .tiff files. Image analysis used a macro in Visilog

5.1 software (Noesis, Gif sur Yvette, France). This allowed for thresholding,

background subtraction, and measurement of the total fluorescence intensity (surface

area, in pixels x mean fluorescence intensity, in grey level) of the signals. In the

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experiment concerning ciprofloxacin exposure, the surface area of diffusion of the DNA

fragments from nucleoids, in number of pixels, was established, for control, 0.012

µg/ml, and 5 µg/ml. Since the data were not normally distributed, as ascertained by the

Kolmogorov-Smirnov test, a non parametric Mann-Whitney U test was performed to

compare between doses.

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RESULTS AND DISCUSSION 268

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Technical implications. Here we present an adapted single-cell diffusion assay, 270

initially used to identify DNA fragmentation in mammalian sperm cells (13, 17, 30), for 271

assessing chromosomal DNA integrity in microorganisms, with relatively small 272

genomes. Intact unfixed microorganisms were immersed in an agarose microgel on a 273

slide, lysed, and stained with a DNA fluorochrome. In higher eukaryotic cells, cells 274

without DNA fragmentation only release DNA loops around a central core, but cells 275

with fragmented DNA produce a large halo of diffusion of DNA spots or fragments. 276

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Given the relatively small genome size of microorganisms, classical fluorochromes such 278

us propidium iodide, DAPI, Hoechst, etc., are not suitable for staining. In order to 279

visualize DNA fragments, it is necessary to use a highly sensitive fluorochrome such us 280

one from the SYBR family. SYBR Gold provides excellent sensitivity and 281

photostability in comparison with other fluorochromes from the same family, giving an 282

accurate visual assessment under the fluorescence microscope (7). Antifading solution 283

was not used since it diminishes the contrast between the small DNA dots and the 284

background. 285

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Validation of the assay: ciprofloxacin treatment and DBD-FISH. Ciprofloxacin is a 287

fluoroquinone that induces DNA double-strand breaks by trapping DNA gyrase and 288

topoisomerase IV on DNA (19). DNA breaks have been shown by several 289

methodologies including viscosity measurements of cell lysates (24, 25). After 290

processing with the Micro-Halomax® kit and SYBR Gold staining, bacteria from 291

untreated control cultures showed nucleoids with DNA loops spreading from a central 292

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core, which corresponds to the residual bacterium, with a compact, microgranular 293

surface, extended peripherally to may branches (Fig. 1a, b). Remarkably, a few 294

bacteria, 0.4%, spontaneously had a very big halo of DNA spots radiating from the 295

residual central core (Fig. 1a, 5a). These images were similar to those visualized in 296

higher eukaryotic cells with fragmented DNA, with the diffusion-based assay (13, 17, 297

30, 36). After treatment with 5 µg/ml ciprofloxacin, all the nucleoids appeared similar 298

to nucleoids with extensive diffusion of DNA spots as observed occasionally in the 299

control cultures. Thus, ciprofloxacin demonstrates that 1) our procedure confidently 300

detects DNA fragmentation, and that 2) images of nucleoids with a big halo of diffusion 301

of DNA spots indicate extremely fragmented DNA. Quantitative analysis of digital 302

images revealed that the mean surface of the nucleoids was 11.5-fold higher than of the 303

untreated control bacteria (Fig. 1d; Table 2). 304

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Interestingly, the effect of ciprofloxacin on DNA integrity was increased with respect to 306

untreated control cultures, at the minimum inhibitory concentration (MIC), i.e., 0.012 307

µg/ml (Fig. 1c). After this low dose, the DNA damage was less and was constant among 308

the bacteria. In fact, the nucleoids appeared more spread, with their average surface 309

being 3.9-fold higher than the untreated control cells (Table 2) and having larger 310

peripheral DNA fragments than after the high dose. As postulated by Drlica et al. (11), 311

ciprofloxacin at low doses like MIC and short incubation times may block growth 312

without killing the cells, suggesting the formation of reversible complexes. At higher 313

doses, like 5 µg/ml, the DNA is extremely fragmented, as here observed, perhaps 314

causing rapid death. The experiment demonstrates the sensitivity and potential value of 315

our procedure for determining the activity of quinolones, both in basic and in clinical 316

research. This approach is currently under investigation in our laboratory. 317

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318

To further confirm the presence of DNA breaks in nucleoids with diffused DNA 319

fragments in control E. coli cultures, the DBD-FISH technique was employed (14, 15). 320

This procedure uses the same microgel as the diffusion assay, allowing simultaneous or 321

sequential visualization of the nucleoids with or without fragmented DNA and labelling 322

of DNA breaks. DBD-FISH is a powerful procedure that involves microgel-embedding, 323

lysis, and incubation with a limited alkaline DNA unwinding step (3, 32). This final step 324

transforms DNA breaks into limited single-stranded DNA (ssDNA) segments generated 325

from the ends of the breaks, which hybridize to fluorescent DNA probes. As DNA 326

breaks increase, more ssDNA is produced, increasing probe hybridization and 327

fluorescence intensity. Fluorescence may be quantified using image analysis software. 328

When hybridizing with a whole genome probe, DNA breaks in the entire genome are 329

assessed. Damage within specific DNA sequence areas may be evaluated by hybridizing 330

specific DNA probes. 331

332

The DBD-FISH procedure was applied to E. coli (Fig. 2) and other microorganisms 333

lysed in the microgel. Nucleoids with diffused spots were strongly labelled, further 334

confirming massive DNA breaks. 335

336

Three kinds of experiments demonstrated the potential of the procedure to determine 337

chromosomal DNA fragmentation: 1) analysis of cells with spontaneous fragmented 338

DNA in culture, 2) reactive oxygen species (ROS) induced DNA damage, and 3) 339

analysis of antibiotic and antifungal agent effects. 340

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Batch cultures. P. mirabilis was incubated in liquid LB medium for 106 h, monitoring 342

turbidity and removing aliquots periodically to determine membrane permeability and 343

DNA fragmentation. Bacteria with fragmented DNA were easily separated from 344

nucleoids without fragmented DNA (Fig. 3a). The frequency of bacteria with DNA 345

fragmentation was established using the Micro-Halomax® kit. This evaluated nucleoids 346

with diffused DNA fragments in 1000-5000 cells per experimental point. Membrane 347

permeability was determined by SYBR Green II and propidium iodide staining. All 348

cells were permeable to SYBR Green II, but propidium iodide is a vital dye, so only 349

cells with membrane permeability, which do not exclude the dye, appear red under the 350

propidium iodide filter set of the microscope. The frequency of propidium iodide 351

permeable cells was established in 1000-5000 microorganisms, per experimental point 352

(Fig. 4). 353

354

The culture changed from exponentially growing to stationary phase after 9 h. The 355

percentage of cells permeable to propidium iodide increased at 48 h from 0.5% to 5%, 356

and then progressively rose through the end of the experiment to 88%. The proportion 357

of bacteria with fragmented DNA significantly increased after 81 h from 0.5-1% to 358

9.5%. It remained constant at 35% from 99 to 103 h and then rose to 52 % 3 h later. 359

360

These results suggest that membrane permeability does not indicate the presence of 361

fragmented DNA, being independent parameters related to different initial targets and 362

not correlated in time. Moreover, the stationary phase seems not to be steady in the 363

frequency of bacteria with fragmented DNA. In the initial period of the stationary 364

phase, the proportion of bacteria with DNA fragmentation did not increase over the 365

exponential phase. The percentage increased later, probably reflecting a progressive 366

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change in the turnover rate between dead and dividing cells. Perhaps with the 367

accumulation of metabolites and the depletion of nutrients, the fraction of cells with 368

fragmented DNA should further increase. 369

370

Hydrogen peroxide treatment. The effect of ROS on DNA integrity was evaluated. 371

Hydrogen peroxide decomposes into hydroxyl radicals (.OH) through catalysis by low 372

valence transition metal ions in a Fenton-Haber-Weiss reaction. These oxidizing agents 373

strongly reacted with macromolecules. .OH attack on DNA results in a variety of base 374

damages and DNA breaks (8, 9). In the only report using the TUNEL assay in bacteria 375

(31), labelling of DNA breaks was detected in exponentially growing cultures of E. coli, 376

after exposure to extremely high doses of H2O2 (86 mM for 30 min). Surprisingly, in 377

stationary phase cultures, even doubling the H2O2 dose (172 mM) did not result in DNA 378

breakage. This suggested that H2O2 does not directly cause DNA breaks, which could be 379

transient intermediates in DNA repair produced by the DNA repair enzymes (31). 380

381

We tested this hypothesis using our DNA fragmentation assay. The percentage of E. 382

coli cells with fragmented DNA was 0.4% and 37.6% in untreated control cells, 383

growing exponentially or in stationary phase, respectively (Fig. 5a, c). Using a lower 384

dose for a shorter time than in the previous report (10mM, 10 min), 100% of nucleoids 385

showed extensively fragmented DNA, either in exponential or in stationary growth 386

phase (Fig 5b, d). This result suggests a higher sensitivity in the microgel-based assay 387

compared with TUNEL in bacteria and illustrates a difficulty with enzymatic 388

procedures for labelling DNA breaks. In the case of TdT, a free 3’-OH group at the 389

terminus of the DNA break is essential as substrate in order to polymerize the 390

nucleotides (10). Attack by agents like H2O2 does not produce “clean” DNA termini, but 391

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rather chemically modified ends, such that direct DNA breaks could be undetectable to 392

enzymes (9). To allow for DNA repair, Exonuclease III removes blocking groups at the 393

3’ terminus (9). Labelling by TdT should therefore be possible. The absence of 394

enzymatic labelling in stationary phase cultures could be explained if end-processing is 395

impaired at this stage. Nevertheless, H2O2-induced DNA breaks are visible with our 396

assay, since it is independent on the chemical nature of the DNA break. Overall, our 397

diffusion assay identifies DNA damage by .OH, both in exponentially and stationary 398

growth phases. 399

400

Ampicillin incubation. To evaluate the influence of ampicillin treatment on 401

chromosomal DNA, exponentially growing cultures of TG1 were exposed to 300 µg/ml 402

ampicillin for 40 min or 24 h. This dose was much higher than the MIC of 3 µg/ml. In 403

contrast to ciprofloxacin, that affects DNA, the cell wall is the primary target for 404

ampicillin; it inhibits peptidoglycan synthesis after binding to penicillin binding 405

proteins and activating autolysins (5, 16). 406

407

Contrary to ciprofloxacin, 40 min treatment with ampicillin barely increased the 408

frequency of cells with fragmented DNA or with appearance of DNA damage. The 409

nucleoids were similar to those from untreated control cells. When incubated for 24 h, 410

the density of bacteria, and correspondent nucleoids, was reduced, but had a uniform 411

background of DNA spots that were probably from spontaneously lysed cells that 412

released the DNA fragments to the medium (Fig. 6). This suggests that cell death 413

initially appears to be independent on DNA damage, but may evolve late in DNA 414

degradation. 415

416

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Amphotericin B incubation in yeast. A presumed PCD has been described in S. 417

cerevisiae, following acidic, oxidative, or osmotic stress, and after ultraviolet exposure 418

(23). This has also been reported for Candida albicans after acetic acid, H2O2, or 419

amphotericin B treatment (28, 29). Apoptotic cells were very significantly increased 420

after 200 min incubation with 4 µg/ml amphotericin B (28). Apoptotic cells were 421

considered those not growing and not permeable to propidium iodide. DNA 422

fragmentation was not assessed. After a dose of 16 µg/ml, practically all Candida cells 423

did not grow, appearing 10% propidium iodide impermeable that were assumed 424

apoptotic, whereas the propidium iodide permeable cells were presumed necrotic. 425

426

An image of C. albicans showing DNA fragmentation is presented in figure 3b. 427

Nevertheless, we assessed the possible induction of DNA fragmentation by 428

amphotericin B in S. cerevisiae. In untreated control cultures, no cells with fragmented 429

DNA were detected in 6000 yeasts. There was no evidence of fragmented DNA with 430

any dose of the antifungal agent when incubated for 3 h. After 24 h incubation with 431

amphotericin B, yeast cells with fragmented DNA were recorded in a dose-dependence 432

manner (Fig. 7). The frequency of cells with fragmented DNA was lower than that with 433

propidium iodide permeable membrane. In fact, with the highest dose assayed, 5% of 434

the cells contained fragmented DNA, whereas 85% were propidium iodide permeable. 435

This decouples membrane permeability from DNA fragmentation as being indicative of 436

death in these microorganisms, at least after treatment with antifungal agents that target 437

to yeast cell membrane, like amphotericin B (4). A substantial and proportional decrease 438

in viability with dose, assayed 48 h after treatment, was demonstrated (Fig. 7), 439

suggesting that DNA fragmentation after amphotericin B treatment is either a rare 440

phenomenon or a late response. 441

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442

Conclusion. The experiments presented here illustrate the ability of the technique, 443

assembled as a kit, to determine the presence of fragmented DNA in microorganisms. 444

Its simplicity, short assay time (50 min) and efficacy, makes this technique useful for 445

the routine determination of DNA fragmentation and intercellular variation. 446

Applications may be extensive in both basic and clinical research. Only a fluorescence 447

microscope is required. Though direct visual identification is quite sharp, the scoring 448

process may be partially automated by adapted image analysis software. This 449

automation could be more complete by integrating a microscope with a motorized plate 450

and focus, a CCD camera for image capture, and image analysis software. This could be 451

useful when scoring many thousands of microorganisms, resembling the flow-cytometer 452

facilities. 453

454

Acknowledgements. Dr. J.L. Fernández , Dr. V. Goyanes and Dr. J. Gosálvez 455

collaborate as scientific advisers of Halotech DNA SL. We are grateful to prof. 456

Christopher de Jonge, from Minnesota University, for the critical reading of the 457

manuscript. This work has been supported by a public grant from the Xunta de Galicia 458

07CSA050916PR and INCITE07PXI916201ES, and by FIS PI061368. 459

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FIGURES 460

461

Figure 1. Images after application of the Micro-Halomax® kit to E. coli cultures. Cells 462

were embedded in an agarose microgel, lysed, and stained with SYBR Gold. a: 463

Nucleoids from control untreated cells, showing spread of DNA loops from a central 464

core. One has highly diffused DNA spots (asterisk). b: A detailed image of an 465

undamaged nucleoid, showing an uneven microgranular surface and multibranched 466

appearance. c: Ciprofloxacin treatment at the MIC dose, 0.012 µg/ml, for 40 min, 467

resulted in DNA fragments. d: These fragments increased after 5 µg/ml treatment; 468

nucleoids showing a big halo of diffusion of DNA spots, similar to the image marked by 469

asterisk in (a). Bar: 5 µm in a, c, d; 2.5 µm in b. 470

471

Figure 2. DBD-FISH detected DNA breaks in nucleoids from control cultures of E. 472

coli. Cells in an agarose microgel were lysed and treated with an alkaline unwinding 473

treatment to transform DNA breaks into restricted single-stranded DNA motifs detected 474

by hybridization with a whole genome probe, Cy-3 labeled (red). The central nucleoid 475

with diffused DNA spots appears intensely labelled, confirming the presence of 476

spontaneous massive DNA breaks (asterisk). Nucleoids without fragmented DNA were 477

visible with DAPI (blue). Bar: 5 µm. 478

479

Figure 3. Images after the application of Micro-Halomax® to cultures of P. mirabilis 480

(a) and C. albicans (b), processed as indicated in the Materials and Methods. Nucleoids 481

without DNA fragmentation released DNA loops around a central core from the residual 482

cell. Otherwise, nucleoids with fragmented DNA were clearly identified by a big halo of 483

DNA spots (asterisks). Bar: 5 µm. 484

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485

Figure 4. Kinetics of the frequency of P. mirabilis cells with fragmented DNA and with 486

propidium iodide permeable membrane. Identification of bacteria with fragmented DNA 487

was performed using the Micro-Halomax® kit, as indicated in the Materials and 488

Methods. 489

490

Figure 5. Exponentially growing cultures from E. coli control cells (a) and exposed to 491

10mM hydrogen peroxide for 10 min (b), evaluated with the diffusion-based assay, 492

using the Micro-Halomax® kit. a: bacterial nucleoids from control cultures only show 493

spreading of DNA loops. Some nucleoids had a big halo of diffused DNA spots, as 494

indicated in the image (asterisk). b: all nucleoids observed after H2O2 treatment reveal a 495

halo of DNA spots. In stationary phase cultures, untreated cells (c) showed similar 496

images to (a), with a higher proportion of background nucleoids with diffused DNA 497

spots (asterisks), whereas those treated with H2O2 (d) were similar to (b). Bar: 5 µm. 498

499

Figure 6. E. coli cultures processed with the Micro-Halomax® kit after ampicillin 500

treatment, 300 µg/ml, 24 h. Nucleoids from residual cells appear more relaxed, 501

accompanied by a dense background of DNA fragments. Bar: 5 µm. 502

503

Figure 7. S. cerevisiae cells with fragmented DNA (right scale), propidium iodide (PI) 504

permeable membrane, and viability (left scale), after incubation for 24 h with increasing 505

doses of amphotericin B.506

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508

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267:836-837. 603

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Gram Negative Bacteria Gram Positive Bacteria

Escherichia coli Enterococcus faecalis

Enterobacter cloacae Staphylococcus aureus

Pseudomonas aeruginosa Streptococcus agalactiae

Proteus mirabilis Streptococcus pyogenes

Salmonella spp.

Stenotrophomonas maltophilia

Acinetobacter haemolyticus

Acinetobacter baumannii

Klebsiella oxytoca

Klebsiella pneumoniae

Citrobacter freundii

Table 1. Bacteria assessed with the Micro-Halomax® kit.

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Dose

(µg/ml) n

Surface Area

(mean ± SD)

Relative Increase

in Surface P

0 150 7411.9 ± 2173.8 - -

0.012 145 29198.8 ± 12747.9 3.9 < 0.0001

5.0 146 85156.8 ± 19301.1 11.5 <0.0001

Table 2. Data of digital image analysis of nucleoids from E. coli treated with

ciprofloxacin, 40 min, in LB broth, and processed with the Micro-Halomax® kit

(n = number of nucleoids examined; surface area is in number of pixels; P =

significance level obtained with Mann-Whitney U test).

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20

40

60

80

100

0 20 40 60 80 100 120

Time (h)

(%)

0

1

2

3

4

5

OD

600

DNA Fragmentation

Membrane Permeability

OD600

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10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

100,00

0 0.5 1 2 4 8 16

[Amphotericin B µµµµg/ml]

% PI Permeable Cells

% Viability

0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

4,00

4,50

5,00

% Cells with Fragmented

DNA

% PI Permeable Cells

% Viability

% Cells with Fragmented DNA

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1 2 DNA FRAGMENTATION IN MICROORGANISMS...

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