Evaluation of Different Methods for Extracting Extracellular DNA from ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2009, p. 5390–5395 0099-2240/09/$08.00⫹0 doi:10.1128/AEM.00400-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 16

Evaluation of Different Methods for Extracting Extracellular DNA from the Biofilm Matrix䌤 Jianfeng Wu and Chuanwu Xi* Department of Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan Received 18 February 2009/Accepted 18 June 2009

The occurrence of high concentrations of extracellular DNA (eDNA) in the extracellular matrices of biofilms plays an important role in biofilm formation and development and possibly in horizontal gene transfer through natural transformation. Studies have been conducted to characterize the nature of eDNA and its potential function in biofilm development, but it is difficult to extract eDNA from the extracellular matrices of biofilms without any contamination from genomic DNA released by cell lysis during the extraction process. In this report, we compared several different extraction methods in order to obtain highly pure eDNA from different biofilm samples. After different extraction methods were explored, it was concluded that using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method. There was no detectable cell lysis when the enzymatic treatment methods were used, but substantial cell lysis was observed when the chemical treatment methods were used. These data suggest that eDNA may bind to other extracellular polymers in the biofilm matrix and that enzymatic treatment methods are effective and favorable for extracting eDNA from biofilm samples. Moreover, randomly amplified polymorphic DNA analysis of eDNA in Acinetobacter sp. biofilms and Acinetobacter sp. genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA.

RAPD analysis, principal-components analysis, and terminal restriction fragment length polymorphism analysis, Steinberger and Holden (33) also characterized eDNA in single- and multiple-species unsaturated biofilm and found that it was different from genomic DNA. However, research is still needed to elucidate the role of eDNA in biofilm structures and in the development and origins of eDNA. In order to further investigate these questions, it is important to extract most of the eDNA of high purity in the biofilm matrix and separate eDNA from other components in the EPSs and from the genomic DNA released during the extraction process. Several methods, such as high-speed centrifugation (2, 33) and membrane filtration (3), have been used to isolate eDNA from biofilm samples. However, these methods may isolate only a portion of the eDNA from biofilm samples. EPSs are composed mainly of high-molecular-weight compounds, including polysaccharides, proteins, and amphiphilic polymers (19, 20), that are secreted by microorganisms into their environment (32). The majority of proteins in the EPSs are bridged by divalent ions, including Ca2⫹ and Mg2⫹, and a small fraction of carbohydrates and nucleic acids are linked to these divalent ions. Under neutral conditions, the carboxyl of protein would become ionized and negative. Through ion interaction, the divalent ions bridge the protein and the cells. In addition, eDNA may be physically or chemically associated with extracellular proteins, polysaccharides, and other polymers in the EPS matrix. The structural assemblage of proteins and polysaccharides in the complex matrix of the EPS might hinder the liberating eDNA from the EPS matrix. Therefore, it is difficult to release eDNA and other materials from the EPS matrix by only vortexing or homogenizing. Additionally, it is necessary to degrade certain components of EPSs in the bio-

A biofilm is a well-organized community of microorganisms that adheres to surfaces and is embedded in the slimy extracellular polymeric substances (EPSs). EPSs are a complex mixture composed of high-molecular-mass polymers (⬎10,000 Da) generated by the bacterial cells, cell lysis and hydrolysis products, and organic matter adsorbed from the substrate. EPSs are involved in the establishment of stable arrangements of microorganisms in biofilms (40), and it recently was found that extracellular DNA (eDNA) is one of the major components of EPSs (7, 31). eDNA plays a very important role in biofilm development (39), and it is believed to be involved in providing substrates for sibling cells, maintaining the three-dimensional structure of biofilms, and enhancing the exchange of genetic materials (18, 31). eDNA has also been found to be accumulated in cultures of several bacterial species and has been postulated as being released by bacterial cells (11, 15, 21, 30). Although it is commonly accepted that eDNA is released mainly from cell lysis (11, 23, 24, 28, 34, 41), several studies have revealed that some other active secretion mechanisms may exist (1, 6, 11, 27). Recent evidence, however, indicates the possibility that eDNA is secreted actively via transport vesicles for the purpose of creating the biofilm matrix (39). Bockelmann et al. found that eDNA formed a defined, network-like spatial structure in the biofilm of an aquatic bacterium and identified that eDNA was not completely identical to genomic DNA by using randomly amplified polymorphic DNA (RAPD) and restriction endonuclease analyses (3). By using

* Corresponding author. Mailing address: Department of Environmental Health Sciences, University of Michigan School of Public Health, 109 Observatory Street, Ann Arbor, MI 48109. Phone: (734) 615-7594. Fax: (734) 936-7283. E-mail: [email protected]. 䌤 Published ahead of print on 26 June 2009. 5390

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TABLE 1. Extractants used in this study Extractant

Property

Function

Reference(s) or source

CER EDTA SDS NaOH

Ion exchange Chelating agent Anionic surfactant Strong alkaline solution

Removes cations Sequesters di- and trivalent metal ions Denatures proteins Ionizes charged groups in proteins and polysaccharides

9, 17 29 29 17, 29

N-Glycanase

Peptide N4-(acetyl-␤-glucosaminyl)-asparagine amidase Glycosyl hydrolase Protein degradation

Releases intact N-linked glycans from glycopeptides and glycoproteins Hydrolyzes ␤-substituted N-acetylglucosamine Nonspecific, subtilisin-related serine protease with a very high sp act

36

Dispersin B Proteinase K

film matrix in order to release eDNAs that may bind to these compounds. In this study, the following extractants were chosen to treat biofilm samples for isolation of eDNA from Acinetobacter sp. strain AC811 biofilm: EDTA and cation-exchange resin (CER) (16), which both have the ability to remove cations from the EPS matrix; sodium dodecyl sulfate (SDS) and NaOH, which are strong denaturants and are used frequently for EPS extraction from various pure and mixed cultures (17, 29); and Nglycanase (glycoprotein degradation hydrolase) (35), dispersin B (biofilm-dispersing glycoside hydrolase) (25), and proteinase K (protein hydrolase). We evaluated the efficiencies of these treatments and their impacts on the quantity and quality of eDNA extracted, and we propose that eDNA may bind to other extracellular polymers in the Acinetobacter biofilm matrix, based on the release of eDNA from the biofilm matrix after such treatments. MATERIALS AND METHODS Bacterial strains, media, and enzymes. Acinetobacter sp. strain AC811 is a derivative of Acinetobacter sp. strain BD413 (which is also known as Acinetobacter sp. strain ADP1 and belongs to the newly described species Acinetobacter baylyi [37]) and is a nonencapsulated strain (12). Acinetobacter sp. strain AC811 was grown in rich medium (Luria-Bertani [LB]) and formed a biofilm in M9 minimal medium supplemented with 0.4% citric acid. Escherichia coli K-12, Pseudomonas aeruginosa PAO1, and Staphylococcus aureus ATCC 25923 were grown on LB plates or in LB broth, and these strains developed biofilms in 1/10 strength of LB broth. Biofilms were grown at room temperature, while batch cultures were grown at 30°C with shaking at 250 rpm. N-Glycanase and proteinase K were obtained from New England Biolabs (Ipswich, MA). Dispersin B was purchased from Kane Biotech, Inc. (Winnipeg, MB, Canada). All other chemicals, unless otherwise specified, were reagent grade or higher and were purchased from Sigma-Aldrich (St. Louis, MO). Biofilm growth and harvesting. Biofilm formation by bacterial strains was achieved using the method described by O’Toole and Kolter (22), with slight modifications. Medium was inoculated from a 1:100 dilution of an overnight culture of tested strains in LB broth and was transferred into the wells of six-well polystyrene plates (catalog no. 0720083LC; Corning Incorporated, Corning, NY) at 3 ml per well. Plates were then incubated at room temperature for 4 days. Biofilm samples were washed with 3 ml of phosphate-buffered saline buffer in each well by gentle pipetting twice, and then biofilm samples in each well were resuspended in 1 ml of 0.9% NaCl solution by scraping of the well bottom and well surface. The biofilm samples of the same strain from different wells were pooled together for eDNA extraction. Extractant treatment. The pooled biofilm samples were homogenized using a homogenizer (TH01; Omni International, Marietta, GA) at 10,000 rpm for 30 s before the measurement of the optical density at 600 nm (OD600). After homogenization, different extractants (summarized in Table 1), including CER (16), EDTA, NaOH, SDS, N-glycanase, dispersin B, and proteinase K, were added to 2 ml of biofilm cell suspensions. Chemical treatments were carried out with 1% CER, 2% EDTA, NaOH at a final pH of 11.0, or 0.01% SDS, and the samples

13, 14 This study

were shaken at 150 rpm for 4 h in the dark at 4°C. A biofilm sample incubated under the same condition but without the addition of any of the extractants was used as the control for the chemical treatments. Enzymatic treatments were carried out as follows. Samples were mixed with 10 ␮g/ml N-glycanase, 20 ␮g/ml dispersin B, or 5 ␮g/ml proteinase K, and the mixtures were incubated at 37°C for 1 h. When a combination of enzymes was applied, the biofilm samples were treated with N-glycanase or dispersin B (or both) at 37°C for 30 min, followed by treatment with proteinase K at 37°C for another 30 min. A biofilm sample without added enzymes was incubated at 37°C for 1 h under the same conditions and was used as the control for the enzymatic treatments. After treatment with different extractants, biofilm samples were filtered through 0.2-␮m polyethersulfone membranes (S623; Whatman, Inc., Florhan Park, NJ); then, half of the eluates (1 ml) was used for quantification of proteins and carbohydrates and cell lysis analysis, while the second half (1 ml) was used for DNA precipitation by the cetyltrimethylammonium bromide (CTAB)-DNA precipitation method (5). Genomic DNA of strain AC811 was extracted from 1 ml of overnight culture by using a MasterPure DNA purification kit (MCD85201; Epicentre Biotechnologies, Madison, WI) according to the protocol described in the manual. In brief, the cells were lysed with tissue and cell lysis solution at 65°C for 15 min, and proteins and cell debris were removed by the addition of MPC protein precipitation reagent and centrifugation at 10,000 ⫻ g for 10 min. The supernatant was used for DNA precipitation by the CTAB-DNA precipitation method (5). The purity of the DNA was checked by determining the ratio of the absorbance at 260 nm to the absorbance at 280 nm. The DNA concentration was measured by determining the absorbance at 260 nm using a spectrophotometer (NanoDrop ND-1000; Thermo Fisher Scientific, Inc., Waltham, MA). Protein concentration analysis. The method of Bradford (4) was applied for analyzing protein concentrations. In brief, 400 ␮l of the filtered sample was mixed gently with 100 ␮l of Bradford dye reagent (catalog no. 500-0006; BioRad, Hercules, CA) in an Eppendorf tube. After 5 min of mixing, 200 ␮l of the mixture was used to measure the absorbance at 595 nm with a microplate reader (Synergy HT; BioTek, Winooski, VT). Bovine serum albumin was used as the standard. Carbohydrate analysis. The anthrone method (10) was applied for carbohydrate determination. A modified method described by Raunkjær et al. (26) was used, and glucose was used as the standard. In brief, 80 ␮l of the sample was mixed with 160 ␮l of anthrone reagent (0.125% anthrone [wt/vol] in 94.5% [vol/vol] H2SO4) with a whirly mixer. Samples were placed in a water bath at 100°C for 14 min and then cooled at 4°C for 5 min. The absorbance at 625 nm was measured using a microplate reader (Synergy HT; BioTek, Winooski, VT). Cell lysis analysis. Cell lysis was measured directly after filtration by using a glucose-6-phosphate dehydrogenase kit (catalog no. G7583-180; Pointe Scientific, Canton, MI) following the protocol in the manufacturer’s manual. Samples treated with NaOH, SDS, EDTA, or proteinase K could not be measured with the kit, since the kit measures only the presence of the glucose-6-phosphate dehydrogenase enzyme. Microscopy and image acquisition. All microscopic observations and image acquisitions were performed with an Olympus IX71 instrument equipped with detectors and filter sets for monitoring propidium iodide (PI) and SYTO-9. Images were obtained with a 100⫻ objective. Homogenized biofilm samples untreated or treated with extractants were stained with Live/Dead BacLight bacterial viability kits (catalog no. L7007; Molecular Probes, Carlsbad, CA) according to the protocol described by the manufacturer and incubated in the dark for 15 min. Bacteria were examined by fluorescence at an excitation wavelength of 485 nm and emission wavelengths of 535 nm (green) and 635 nm (red).

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The percentages of live and dead bacteria for each sample were calculated from three images. RAPD analysis of Acinetobacter sp. strain AC811 eDNA and genomic DNA. RAPD analysis was performed with Acinetobacter sp. strain AC811 genomic DNA and eDNA according to the method described by Franklin et al. and Venieri et al. (8, 38). Two different oligonucleotide primers (F1 and T7) (8) were used. Each 25-␮l reaction contained 40 ng of the DNA template, 40 pmol of oligonucleotide, 1 U GoTaq polymerase (Promega), 250 mM (each) deoxynucleoside triphosphates, and 3 ␮M MgCl2 in PCR buffer. Amplification was done with a Mastercycler gradient machine (Eppendorf, Westbury, NY) by denaturing at 95°C for 5 min, followed by 40 cycles at 94°C for 1 min, 36°C for 1 min, and 72°C for 2 min and a final extension at 72°C for 10 min. The RAPD products were separated by gel electrophoresis in a 2% agarose gel. The gel was subsequently stained with SYBR green I (Cambrex, Rockland, ME) and photodocumented using a UV transilluminator (Fotodyne, Inc., Hartland, WI). In order to test whether chemical treatments (e.g., SDS and NaOH) could modify DNA chemistry differently from enzyme-based treatments and exert bias on PCR amplification, eDNAs extracted with enzymatic treatments were incubated with SDS or NaOH and then purified and precipitated using the CTAB-DNA precipitation method. No changes in the RAPD patterns were observed from the additional chemical incubation steps (data not shown). Unique RAPD bands (B1, B2, B3, and B4) (see Fig. 3) amplified from eDNA were excised from a 2% agarose gel, purified using a Wizard SV gel and PCR clean-up system (Promega, Madison, WI), religated to a pGEM-T easy cloning vector (Promega, Madison, WI), and transformed into calcium chloride-competent E. coli JM109 cells (Promega, Madison, WI) according to the manufacturer’s instructions. Colonies containing the corresponding cloned bands were selected for DNA sequencing at the University of Michigan’s DNA Sequencing Core. All sequences were aligned with the Acinetobacter sp. strain ADP1 genome sequence in the NCBI database (http://www.ncbi.nlm.nih.gov).

RESULTS Dispersion of biofilm matrix by treatments with different extractants. The 4-day biofilms of Acinetobacter sp. strain AC811 cultivated in six-well plates were pretreated with different extractants as described in Materials and Methods. After pretreatment, the Acinetobacter sp. strain AC811 biomass was stained with PI and SYTO-9 dyes and examined with a fluorescence microscope. The controls for the chemical treatments and the enzymatic treatments appeared similar to those for the homogenized biofilm control (data not shown). Most cells from these controls showed green fluorescence (⬎85%), indicating that the membranes of most cells were intact (Fig. 1A). Most cells from samples treated with CER and various enzymes also showed green fluorescence (⬎85%) (Fig. 1B, F, G, and H), and the cells from CER-treated samples and all control samples (including the homogenized biofilm control and the controls for chemical treatments and enzymatic treatments) remained clustered together, although the samples were homogenized prior to staining and microscope observation. Cells from enzyme-treated samples were highly dispersed, and most of them were present as single cells (⬎90%) (Fig. 1F, G, and H). For samples treated with chemical extractants (EDTA, SDS, and NaOH), most cells showed red fluorescence (stained with PI, indicating that membranes of cells were impaired), while there were still a large number of clustered cells (⬎95%) (Fig. 1C, D, and E). These data indicate that enzymatic treatment can disperse the biofilm matrix very well, without damaging cell membranes. A glucose-6-phosphate dehydrogenase assay revealed that there was no detectable enzyme activity in eluates of biofilm samples after treatment with CER and enzymes (N-glycanase and dispersin B) (data not shown), indicating no obvious cell lysis due to these treatments. These

FIG. 1. Micrographs of biofilm samples treated with different extractants and stained with PI and SYTO-9. Biofilm samples without treatment (A) or treated with CER (B), EDTA (C), SDS (D), NaOH (E), N-glycanase (F), dispersin B (G), or proteinase K (H) are included. All biofilm samples were homogenized before microscopic observation. Bar, 20 ␮m.

data are consistent with results obtained with the Live/Dead staining assay. Extraction of EPS from Acinetobacter sp. strain AC811 biofilms. After pretreatment with different extractants and filtration through 0.2-␮m polyethersulfone membranes, the concentrations of proteins and carbohydrates in the eluates were determined. The CTAB-DNA isolation method was used to precipitate DNA from each eluate, and the concentration of extracted DNA was also determined. The concentrations of protein, carbohydrates, and DNAs from the biofilm samples treated with different extractants are summarized in Table 2. The concentrations of these compounds extracted from the controls for chemical and enzymatic treatments are in the same range, shown as the control in Table 2. The control treatment obtained the least amount of eDNA from Acinetobacter sp. strain AC811 biofilms. The CER treatment resulted in a 1.4fold increase in the amount of eDNA extracted compared to

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TABLE 2. Yields of eDNA, proteins, and carbohydrates from eluates of 4-day Acinetobacter sp. strain AC811 biofilm matrix after treatments with different extractants Treatment

Control CER (100 mg/OD600) EDTA (1%) SDS (0.1%) NaOH (pH 11.0) N-Glycanase Dispersin B Proteinase K a b

eDNA ratio (treatment/control)

eDNA (␮g)a

Proteins (␮g)a

Carbohydrates (␮g)a

0.24 ⫾ 0.03

42.54 ⫾ 8.03

22.10 ⫾ 2.89

1.0

0.33 ⫾ 0.05 3.45 ⫾ 0.60 87.72 ⫾ 14.30 135.19 ⫾ 13.51

34.60 ⫾ 7.54 40.97 ⫾ 6.23 118.67 ⫾ 14.14 155.65 ⫾ 12.81

33.21 ⫾ 5.40 35.07 ⫾ 3.31 88.39 ⫾ 3.31 96.06 ⫾ 8.68

1.4 14.5 368.4 567.8

12.59 ⫾ 3.12 14.44 ⫾ 2.40 7.86 ⫾ 3.23

67.55 ⫾ 8.11 66.12 ⫾ 3.27 NAb

25.95 ⫾ 5.32 26.42 ⫾ 3.68 27.43 ⫾ 5.50

52.9 60.7 33.0

The values are given as ␮g/OD600 obtained from 1 ml of biofilm samples and are means ⫾ standard deviations from three replicates. NA, not applicable.

that from the control treatment. When EDTA was used as an extractant, a 14.5-fold increase in the amount of eDNA was extracted. The enzyme (N-glycanase, dispersin B, and proteinase K) treatment can yield as high as 60 times the amount of eDNA without a substantial increase in protein and carbohydrate in the eluates. The highest eDNA yield (368- to 567-fold) was obtained when SDS or NaOH served as the extractant (Table 2). However, large amounts of proteins and carbohydrates were also detected in the eluates when this treatment was used. In addition, the Live/Dead staining revealed some damage to the cell membrane by these two treatments (Fig. 1), suggesting that the highest yield of eDNA may come from the genomic DNA released during the treatment process. In order to investigate the efficiency of eDNA extraction by use of enzymatic treatment methods, Acinetobacter sp. strain AC811 biofilm samples were treated with combinations of various enzymes. Single-enzyme treatments with N-glycanase, dispersin B, or proteinase K can all yield more eDNA than the control, and dispersin B treatment is the best among them (Fig. 2). The combination of proteinase K with N-glycanase or dispersin B can increase the eDNA yield, and the dispersin B and proteinase K combination yields the largest amount of eDNA from Acinetobacter sp. strain AC811 biofilms (30-fold increase compared to the yield obtained with the control treatment). No significant increase in eDNA extraction was observed for AC811 biofilm samples treated with all three enzymes compared to that for samples treated with the combination of dispersin B and proteinase K (Fig. 2). Isolation of eDNA from biofilm samples from E. coli K-12, P. aeruginosa PAO1, and S. aureus ATCC 25923. To test the efficiency of enzymatic treatment methods for extraction of eDNA from other biofilm samples, biofilm samples from E. coli K-12, P. aeruginosa PAO1, and S. aureus ATCC 25923 were treated in the same manner, with individual enzymes or combinations of proteinase K, N-glycanase, and dispersin B as described above, and eDNAs from each treatment were isolated and quantified. The increases in eDNA yield varied for biofilms formed by different strains with different enzymatic treatments (Fig. 2). Regarding the biofilm sample formed by E. coli K-12, the N-glycanase treatment yielded the largest amount of eDNA among the single-enzyme treatments, a threefold increase from the yield obtained with the control

treatment (Fig. 2). The dispersin B treatment did not significantly increase the yield of eDNA. For the biofilms developed by P. aeruginosa PAO1 and S. aureus ATCC 25923, the Nglycanase treatment and the proteinase K treatment yielded the largest amounts of eDNA among the single-enzyme treatments, a 4-fold and an 11-fold increase from the yield obtained with the control treatment, respectively. Data also showed that when combination treatments were performed, the combination of N-glycanase and proteinase K treatment yielded the largest amount of eDNA from the biofilm matrixes of all tested strains (Fig. 2). There was no detectable cell lysis by use of the glucose-6-phosphate dehydrogenase assay during the procedure of eDNA extraction with enzymes for any of the strains, except that the proteinase K treatment was not tested (data not shown).

FIG. 2. Yields of eDNA isolated from 4-day biofilms after biofilm samples were treated with different enzymes and combinations. Biofilms formed by Acinetobacter sp. (A. baylyi) strain AC811, E. coli K-12, P. aeruginosa PAO1, and S. aureus ATCC 25923 were used for eDNA extraction. Biofilm samples were treated by filtration (ck, control); with N-glycanase, dispersin B, or proteinase K; or with various combinations (G ⫹ K, combination of N-glycanase and proteinase K; D ⫹ K, combination of dispersin B and proteinase K; G ⫹ D ⫹ K, combination of all three enzymes). Bars represent means and standard deviations from three replicates. Statistical analysis was done by using Student’s t test. Different letters above the bars indicate significant differences (P ⬍ 0.05).

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homologs of previously reported genes of unknown function, except ACIAD0551 (peptidoglycan [murein] turnover, recycling [MultiFun 1.7.34]). DISCUSSION

FIG. 3. Comparative RAPD analysis, done using two sets of primers (A, primer F1; B, primer T7), of Acinetobacter sp. strain AC811 genomic DNA (lane 10) and eDNA obtained from biofilm samples by using the following different extractants: control (lane 1), CER (lane 2), EDTA (lane 3), SDS (lane 4), NaOH (lane 5), N-glycanase (lane 6), dispersin B (lane 7), proteinase K (lane 8), and a combination of dispersin B and proteinase K (lane 9). Unique bands (B1 to B4, marked with arrows) were excised and sequenced.

RAPD analysis of eDNAs extracted from Acinetobacter sp. strain AC811 biofilms with different treatments. The RAPD analysis with two different sets of primers (F1 and T7) was performed on Acinetobacter sp. strain AC811 genomic DNA and eDNAs extracted from Acinetobacter sp. strain AC811 biofilms pretreated with different extractants. The patterns of amplified fragments from Acinetobacter sp. strain AC811 genomic DNA and eDNA extracted with EDTA, SDS, and NaOH treatments were similar (Fig. 3) but differed substantially from the patterns from eDNA extracted with CER treatment and all enzymatic treatments. This similarity and difference were the same using both sets of primers. The unique bands amplified from eDNA (B1, B2, B3, and B4) (Fig. 3) were cloned and sequenced. The sequence of B1 (364 bp, aligned with nucleotides 542584 to 542947 in the Acinetobacter sp. strain ADP1 genome) is located in the partial coding regions of ACIAD0550 and ACIAD0551; the sequence of B2 (888 bp, aligned with nucleotides 566224 to 567111) is located in the coding regions of ACIAD0576 and ACIAD0577; the sequence of B3 (554 bp, aligned with nucleotides 566557 to 567111) is located in the coding regions of ACIAD0576 and ACIAD0577; and the sequence of B4 (210 bp, aligned with nucleotides 566349 to 566559) is located in the coding region of ACIAD0576. All of these annotated open reading frames are

In this report, we compared the efficiencies of treatments with CER, chemicals (EDTA, SDS, and NaOH), and enzymes (N-glycanase, dispersin B, and proteinase K) for extraction of eDNA from Acinetobacter sp. strain AC811 biofilms. The enzymatic treatments were found to be superior to the other methods in terms of eDNA yield and lack of detectable cell lysis during the extraction process. The eDNAs extracted using enzymatic methods were 60 to 190 times higher than those from the control (Table 2 and Fig. 2), and no observable cell lysis was found during extraction (indicated by SYTO-9/PI staining and glucose-6-phosphate dehydrogenase assay) (Fig. 1). The data also suggest that a large portion of the eDNA may bind to extracellular polysaccharides, proteins, and other polymers in the biofilm matrix. eDNA may be divided into two portions, free eDNA and bound eDNA. The enzymatic treatments can degrade the matrix and release the bound eDNA. It is important to extract this portion of bound eDNA when we try to understand the composition and function of eDNA in biofilm development. Although SDS and NaOH treatments yielded larger amounts of eDNA than did the control treatment (360 to 560 times more than that from the control), Live/Dead staining and microscopic observation suggested that a large number of cells had membrane damage during the extraction process (Fig. 1). This may affect the eDNA quality due to the contamination of genomic DNA released from the membrane-impaired cells during the extraction process. The data also suggested that the enzymatic treatment methods for eDNA extraction were suitable for biofilms developed by other bacterial strains, including P. aeruginosa PAO1, E. coli K-12, and S. aureus ATCC 25923 tested in this study (Fig. 2). Treatments with proteinase K, dispersin B, and N-glycanase yielded different amounts of eDNA from each of these strains (Fig. 2), which may be due to different compositions of extracellular polymers in the biofilm matrixes. However, the combination of N-glycanase and proteinase K treatments yielded significantly increased amounts of eDNA from the biofilm matrixes of all four tested bacterial strains. Therefore, it is recommended that this extraction method be used for studying eDNA from biofilms developed by different strains. Previous studies have suggested that eDNAs are released mainly from cell lysis (11, 23, 24, 28, 34, 41), but several studies also revealed that some active secretion mechanisms may exist (1, 6, 11, 27). It is postulated that eDNA originates from genomic DNA and that genomic DNA is released into the biofilm matrix via cell lysis and/or active secretion. However, there may be alterations to the DNA during and/or after it is released, e.g., breakdown or degradation of genomic DNA. The RAPD analysis revealed different patterns of DNA bands amplified from eDNA extracted with CER and enzymatic treatment methods (Fig. 3) compared to patterns of bands amplified from genomic DNA and eDNA extracted using EDTA, SDS, and NaOH treatments. Sequences of unique RAPD bands amplified from eDNA are identical to the Acinetobacter sp. strain ADP1 genome sequences and are located in

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METHODS FOR EXTRACTING eDNA FROM THE BIOFILM MATRIX

the same region of the genome. These data suggest that eDNA in the biofilm matrix originated from the genomic DNA. The biological meaning of abundance of a specific region of genomic DNA in eDNA deserves further investigation. Genomic DNA released from cell lysis may undergo physical or chemical changes after being released from cell lysis. If actively secreted genomic DNA is part of the eDNA in the biofilm matrix, only certain regions of genomic DNA may be secreted. Therefore, further investigations of the extent of cell lysis and the contribution of active DNA secretion to eDNA in the biofilm matrix (and the extent of each) are warranted. These differences may not be detected if eDNA is contaminated with excessive amounts of genomic DNA from cell lysis during the extraction process, which was the case in this study when the EDTA, SDS, and NaOH treatment methods were used for eDNA extraction. In conclusion, the difference in eDNA yield obtained using different enzymatic treatments suggests that eDNA and other polymers (including proteins and polysaccharides) bind to each other and form a network structure to support biofilm growth in a three-dimensional manner. The enzymatic treatment used for eDNA extraction can extract both free eDNA and bound eDNA in the biofilm matrix and can prevent contamination of genomic DNA released from cell lysis during the extraction process by other methods. Furthermore, the combination of N-glycanase and proteinase K treatments is recommended to extract eDNA from the biofilm matrix. For particular strains of interest, prior testing with different combinations of enzymes may be necessary to optimize the method. This method should be very useful for further characterization of the composition of eDNA and the role of eDNA in biofilm development. REFERENCES 1. Abajy, M. Y., J. Kopec, K. Schiwon, M. Burzynski, M. Doring, C. Bohn, and E. Grohmann. 2007. A type IV-secretion-like system is required for conjugative DNA transport of broad-host-range plasmid pIP501 in gram-positive bacteria. J. Bacteriol. 189:2487–2496. 2. Allesen-Holm, M., K. B. Barken, L. Yang, M. Klausen, J. S. Webb, S. Kjelleberg, S. Molin, M. Givskov, and T. Tolker-Nielsen. 2006. A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol. Microbiol. 59:1114–1128. 3. Bockelmann, U., A. Janke, R. Kuhn, T. R. Neu, J. Wecke, J. R. Lawrence, and U. Szewzyk. 2006. Bacterial extracellular DNA forming a defined network-like structure. FEMS Microbiol. Lett. 262:31–38. 4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. 5. Corinaldesi, C., R. Danovaro, and A. Dell’Anno. 2005. Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments. Appl. Environ. Microbiol. 71:46–50. 6. Draghi, J. A., and P. E. Turner. 2006. DNA secretion and gene-level selection in bacteria. Microbiology 152:2683–2688. 7. Flemming, H. C., and J. Wingender. 2001. Relevance of microbial extracellular polymeric substances (EPSs)—part II: technical aspects. Water Sci. Technol. 43:9–16. 8. Franklin, R. B., D. R. Taylor, and A. L. Mills. 1999. Characterization of microbial communities using randomly amplified polymorphic DNA (RAPD). J. Microbiol. Methods 35:225–235. 9. Frolund, B., R. Palmgren, K. Keiding, and P. H. Nielsen. 1996. Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Res. 30:1749–1758. 10. Gaudy, E., and R. S. Wolfe. 1962. Composition of an extracellular polysaccharide produced by Sphaerotilus natans. Appl. Microbiol. 10:200–205. 11. Hamilton, H. L., N. M. Dominguez, K. J. Schwartz, K. T. Hackett, and J. P. Dillard. 2005. Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system. Mol. Microbiol. 55:1704–1721. 12. Juni, E., and A. Janik. 1969. Transformation of Acinetobacter calco-aceticus (Bacterium anitratum). J. Bacteriol. 98:281–288. 13. Kaplan, J. B., C. Ragunath, N. Ramasubbu, and D. H. Fine. 2003. Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous ␤-hexosaminidase activity. J. Bacteriol. 185:4693–4698. 14. Kaplan, J. B., C. Ragunath, K. Velliyagounder, D. H. Fine, and N. Rama-

15. 16.

17.

18.

19. 20. 21.

22.

23.

24.

25.

26.

27.

28.

29.

30. 31. 32.

33. 34.

35.

36.

37.

38.

39. 40.

41.

5395

subbu. 2004. Enzymatic detachment of Staphylococcus epidermidis biofilms. Antimicrob. Agents Chemother. 48:2633–2636. Lorenz, M. G., and W. Wackernagel. 1994. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev. 58:563–602. Martin-Cereceda, M., F. Jorand, A. Guinea, and J. C. Block. 2001. Characterization of extracellular polymeric substances in rotating biological contractors and activated sludge flocs. Environ. Technol. 22:951–959. McSwain, B. S., R. L. Irvine, M. Hausner, and P. A. Wilderer. 2005. Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge. Appl. Environ. Microbiol. 71:1051–1057. Molin, S., and T. Tolker-Nielsen. 2003. Gene transfer occurs with enhanced efficiency in biofilms and induces enhanced stabilisation of the biofilm structure. Curr. Opin. Biotechnol. 14:255–261. Neu, T. R. 1996. Significance of bacterial surface-active compounds in interaction of bacteria with interfaces. Microbiol. Rev. 60:151–166. Neu, T. R., and J. R. Lawrence. 1999. Lectin-binding analysis in biofilm systems. Methods Enzymol. 310:145–152. Nielsen, K. M., P. J. Johnsen, D. Bensasson, and D. Daffonchio. 2007. Release and persistence of extracellular DNA in the environment. Environ. Biosafety Res. 6:37–53. O’Toole, G. A., and R. Kolter. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28:449–461. Patton, T. G., K. C. Rice, M. K. Foster, and K. W. Bayles. 2005. The Staphylococcus aureus cidC gene encodes a pyruvate oxidase that affects acetate metabolism and cell death in stationary phase. Mol. Microbiol. 56: 1664–1674. Qin, Z., Y. Ou, L. Yang, Y. Zhu, T. Tolker-Nielsen, S. Molin, and D. Qu. 2007. Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis. Microbiology 153:2083–2092. Ramasubbu, N., L. M. Thomas, C. Ragunath, and J. B. Kaplan. 2005. Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans. J. Mol. Biol. 349:475–486. Raunkjær, K., T. Hvitved-Jacobsen, and P. H. Nielsen. 1994. Measurement of pools of protein: carbohydrate and lipid in domestic wastewater. Water Res. 28:12. Renelli, M., V. Matias, R. Y. Lo, and T. J. Beveridge. 2004. DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential. Microbiology 150:2161–2169. Rice, K. C., J. B. Nelson, T. G. Patton, S. J. Yang, and K. W. Bayles. 2005. Acetic acid induces expression of the Staphylococcus aureus cidABC and lrgAB murein hydrolase regulator operons. J. Bacteriol. 187:813–821. Sheng, G. P., H. Q. Yu, and Z. Yu. 2005. Extraction of extracellular polymeric substances from the photosynthetic bacterium Rhodopseudomonas acidophila. Appl. Microbiol. Biotechnol. 67:125–130. Smithies, W. R., and N. E. Gibbons. 1955. The deoxyribose nucleic acid slime layer of some halophilic bacteria. Can. J. Microbiol. 1:614–621. Spoering, A. L., and M. S. Gilmore. 2006. Quorum sensing and DNA release in bacterial biofilms. Curr. Opin. Microbiol. 9:133–137. Staudt, C., H. Horn, D. C. Hempel, and T. R. Neu. 2004. Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms. Biotechnol. Bioeng. 88:585–592. Steinberger, R. E., and P. A. Holden. 2005. Extracellular DNA in single- and multiple-species unsaturated biofilms. Appl. Environ. Microbiol. 71:5404–5410. Steinmoen, H., E. Knutsen, and L. S. Havarstein. 2002. Induction of natural competence in Streptococcus pneumoniae triggers lysis and DNA release from a subfraction of the cell population. Proc. Natl. Acad. Sci. USA 99: 7681–7686. Suzuki, T., and W. J. Lennarz. 2003. Hypothesis: a glycoprotein-degradation complex formed by protein-protein interaction involves cytoplasmic peptide: N-glycanase. Biochem. Biophys. Res. Commun. 302:1–5. Tarentino, A. L., and T. H. Plummer, Jr. 1994. Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Methods Enzymol. 230:44–57. Vaneechoutte, M., D. M. Young, L. N. Ornston, T. De Baere, A. Nemec, T. Van Der Reijden, E. Carr, I. Tjernberg, and L. Dijkshoorn. 2006. Naturally transformable Acinetobacter sp. strain ADP1 belongs to the newly described species Acinetobacter baylyi. Appl. Environ. Microbiol. 72:932–936. Venieri, D., A. Vantarakis, G. Komninou, and M. Papapetropoulou. 2004. Differentiation of faecal Escherichia coli from human and animal sources by random amplified polymorphic DNA-PCR (RAPD-PCR). Water Sci. Technol. 50:193–198. Whitchurch, C. B., T. Tolker-Nielsen, P. C. Ragas, and J. S. Mattick. 2002. Extracellular DNA required for bacterial biofilm formation. Science 295:1487. Wolfaardt, G. M., J. R. Lawrence, R. D. Robarts, and D. E. Caldwell. 1998. In situ characterization of biofilm exopolymers involved in the accumulation of chlorinated organics. Microb. Ecol. 35:213–223. Yang, S. J., K. C. Rice, R. J. Brown, T. G. Patton, L. E. Liou, Y. H. Park, and K. W. Bayles. 2005. A LysR-type regulator, CidR, is required for induction of the Staphylococcus aureus cidABC operon. J. Bacteriol. 187:5893–5900.