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Exogenous Lytic Activity of SPN9CC Endolysin Against Gram-Negative Bacteria
Exogenous Lytic Activity of SPN9CC Endolysin Against Gram-Negative Bacteria
Journal of Microbiology and Biotechnology. 2014. Jun, 24(6): 803-811
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : March 13, 2014
  • Accepted : March 24, 2014
  • Published : June 30, 2014
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About the Authors
Jeong-A Lim
Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, Center for Agricultural Biomaterials and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea
Hakdong Shin
Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, Center for Agricultural Biomaterials and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea
Sunggi Heu
Microbial Safety Division, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
Sangryeol Ryu
Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, Center for Agricultural Biomaterials and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea
sangryu@snu.ac.kr

Abstract
Concerns over drug-resistant bacteria have stimulated interest in developing alternative methods to control bacterial infections. Endolysin, a phage-encoded enzyme that breaks down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle, is reported to be effective for the control of bacterial pathogenic bacteria. Bioinformatic analysis of the SPN9CC bacteriophage genome revealed a gene that encodes an endolysin with a domain structure similar to those of the endolysins produced by the P1 and P22 coliphages. The SPN9CC endolysin was purified with a C-terminal oligo-histidine tag. The endolysin was relatively stable and active over a broad temperature range (from 24℃ to 65℃). It showed maximal activity at 50℃, and its optimum pH range was from pH 7.5 to 8.5. The SPN9CC endolysin showed antimicrobial activity against only gram-negative bacteria and functioned by cutting the glycosidic bond of peptidoglycan. Interestingly, the SPN9CC endolysin could lyse intact gram-negative bacteria in the absence of EDTA as an outer membrane permeabilizer. The exogenous lytic activity of the SPN9CC endolysin makes it a potential therapeutic agent against gram-negative bacteria.
Keywords
Introduction
Over the last decade, a dramatic increase in the prevalence of antibiotic resistance has occurred in several significant pathogens [11] . It is difficult to develop new classes of antibiotics to control antibiotic-resistant bacteria, and alternative therapeutic reagents or methods are required [3 , 21] .
Bacteriophage endolysin is an enzyme that targets bacterial peptidoglycan. Generally, bacteriophages, which have double-stranded DNA, encode two proteins for host lysis, holin and endolysin. In the last step of the lytic bacteriophage life cycle, endolysin passes through a cytoplasmic membrane pore formed by holin to reach its target, peptidoglycan. Endolysin hydrolyzes peptidoglycan by cleaving a glycosidic bond, peptide bond, or amide bond, and host bacteria are then lysed by osmotic pressure. Finally, progeny virions can be released [1 , 23 , 27 , 34] . In the case of gram-negative bacteria, additional supporting proteins (Rz/Rz1-like proteins) may be needed for full lysis [40] .
Purified endolysins have the potential to serve as a new class of therapeutic agents against gram-positive bacteria by acting as exolysins because they make direct contact with the cell wall of gram-positive bacteria [2] . Endolysins have a number of advantages, including bactericidal activity, being made from a naturally occurring material, and effectiveness against antibiotic-resistant bacteria [8] . The target specificity of endolysin especially renders it a promising antibiotic substitute, because endolysin is not toxic to the eukaryotic cell and does not disrupt environmental microorganisms [8 , 23] . In many studies, endolysins have been shown to have antimicrobial activity against pathogens such as Streptococcus pneumoniae , Bacillus anthracis , B. subtilis , Staphylococcus aureus , Lactobacillus fermentum , Listeria monocytogenes , Enterococcus faecalis , and Clostridium perfringens [9 , 16 , 22 , 31 , 36 , 38 , 42 - 44] .
However, the activity of endolysin against gram-negative bacteria is limited because the outer membrane of gramnegative bacteria acts as a physical barrier and prevents external endolysin from accessing the peptidoglycan [6 , 8] . Thus, most studies have focused on the activity of endolysin against gram-positive bacteria, and few have examined the activity of endolysin produced from bacteriophages that infect gram-negative bacteria such as Pseudomonas aeruginosa , Klebsiella spp., Acinetobacter baumannii , and Enterobacteria [4 , 15 , 19 , 27 , 28 , 32] . To overcome this limitation, new strategies of enhancing outer membrane permeability using chelating agents and high hydrostatic pressure have been reported [5 , 30] . Recent reports also describe a structurally engineered endolysin that targets gram-negative pathogens [24 , 25] , suggesting that phage endolysins might be used as therapeutic agents not only for gram-positive bacteria but also for gram-negative bacteria.
Bacteriophage SPN9CC was isolated from a commercially processed broiler skin sample collected from the traditional market, using Salmonella Typhimurium as host bacteria [39] . Previous study of phage SPN9CC showed that the host lysis gene cluster of this phage consisted of holin, endolysin, and genes encoding Rz/Rz1-like proteins, and bioinformatic analysis of its genome (GenBank Accession No. JF900176) identified a lysis gene cluster that is very similar to those of the P22-like phage ST104 and the E. coli K-12 DLP12 prophage. Expression analysis of these genes revealed that the proteins encoded by the lysis cluster genes were critical for the lysis of Salmonella and Escherichia coli [39] .
In this study, an endolysin from bacteriophage SPN9CC that targets Salmonella was purified, and its lytic activity against gram-negative bacteria was analyzed. Our results suggest that it has potential for the control of gramnegative pathogens. Furthermore, the specific cleavage site of the peptidoglycan was identified. Information obtained from studying the SPN9CC endolysin will be useful in the development of endolysin-based biocontrol agents with activity against multiple gram-negative pathogens.
Materials and Methods
- Purification of Recombinant SPN9CC Endolysin
Plasmids and oligonucleotides used to clone and amplify the SPN9CC endolysin.
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Plasmids and oligonucleotides used to clone and amplify the SPN9CC endolysin.
Bacteriophage SPN9CC DNA was prepared as previously described [35] . To express a C-terminal oligo-histidine-tagged endolysin, the endolysin-encoding gene was amplified by polymerase chain reaction and cloned into pET29b. The plasmids and primers are listed in Table 1 . E. coli BL21 (DE3) was transformed with the pET29-lys plasmid. The expression of the recombinant SPN9CC endolysin was induced by the addition of 1 mM IPTG when the optical density (600 nm) of the E. coli culture harboring the recombinant plasmid reached 0.6. After an incubation for an additional 4 h, the bacterial cells were harvested and suspended with buffer containing 50 mM sodium phosphate (pH 7.0) and 300 mM NaCl and were then disrupted by sonication (Bioruptor; Diagenode, Denville, NJ, USA). Recombinant SPN9CC endolysin was purified by affinity chromatography using nickelnitriloacetic acid agarose resin (Qiagen, Hilden, Germany). The purified protein was stored at -80℃ in 50 mM sodium phosphate buffer (pH 7.0) containing 300 mM NaCl and 30% glycerol.
- Bioinformatic Analysis
The functional domain analysis of the SPN9CC endolysin was conducted using the NCBI Conserved Domain Database (CDD) [26] and the InterProScan database [46] . The transmembrane helix was predicted using the program TMHMM ver. 2.0 [17] . The protein hydrophobicity was plotted using CLC Main Workbench ver. 6.9.1 (CLC Bio, Denmark) with the Kyte-Doolittle scale [18] .
- Confirmation of Antimicrobial Activity
An E. coli DH5α broth culture in the early exponential phase was treated with 100 mM EDTA as described previously [15 , 20] . Different amounts of SPN9CC endolysin (0.1, 0.5, 1, or 5 μg) were mixed with the EDTA-treated DH5α suspension (1 ml). The antimicrobial activity of recombinant endolysin could be confirmed by measuring the reduction in the optical density (600 nm) of the cells. The same volume of endolysin storage buffer was used as a negative control.
In an alternative assay, an exponentially growing E. coli MG1655 culture was washed and diluted 100-fold in phosphate buffered saline. SPN9CC endolysin (10 or 30 μg) was added to 100 μl of cells with EDTA (final concentrations of EDTA were 1 or 5 mM). As a negative control, the same volume of buffer was used instead of endolysin or EDTA. After a 1 or 2 h reaction, the residual viable cell numbers were measured. All of the experiments were repeated at least three times. The error bars represent the standard deviations.
- Effects of pH and Temperature on the Enzymatic Activity of SPN9CC Endolysin
To verify the optimum pH for bacterial lysis activity, the enzymatic reaction was performed at different pH values using the JBScreen pH-2D broad-range pH buffer (Jena Bioscience, Germany). The relative lysis activity was calculated by combining the results obtained with six different buffers ranging from pH 4 to 10 (SSG buffer, succinic acid: sodium dihydrogen phosphate: glycine = 2:7:7; CHC buffer, citric acid:HEPES:CHES = 2:3:4; MIB buffer, malonic acid:imidazole:boric acid = 2:3:3) or with three different types of buffers ranging in pH from 4 to 9 (SAB buffer, sodium acetate:ADA:bicine = 1:1:1; MMT buffer, L -malic acid: MES:tris = 1:2:2; SBG buffer, sodium tartrate dehydrate:bistris: glycylglycine = 3:2:2). To determine the optimum temperature for the SPN9CC endolysin, the antimicrobial activity assay was performed at different temperatures (24℃, 30℃, 37℃, 45℃, 50℃, 55℃, 60℃, or 65℃) for 3 min. The relative lysis activity was calculated in proportion to the highest activity. To test the thermostability of the SPN9CC endolysin, aliquots (0.5 μg) were incubated at different temperatures (4℃, 24℃, 37℃, 45℃, 55℃, or 65℃). After 3 min, 10 min, 30 min, 1 h, or 2 h, the residual lysis activity was compared. In all of the assays, the lysis activity was detected by measuring the optical density before and after the addition of the endolysin to EDTA-treated E. coli DH5α cells. The experiments were performed three times and the standard deviations were represented by error bars.
- Antimicrobial Spectrum of the SPN9CC Endolysin
A total of eight gram-positive strains were grown to earlyexponential phase and washed with 50 mM Tris-HCl (pH 8.0); then, 1 ml of each cell suspension was used in the enzymatic assay. A total of 23 gram-negative strains, including Salmonella enterica (10 strains) and E. coli (6 strains), were grown to early-exponential phase and treated with 100 mM EDTA. The SPN9CC endolysin (0.5 μg) was added to the prepared bacterial cells, and the enzyme reaction was maintained for 5 min. The relative lysis activity was calculated from the difference in the optical densities at 600 nm between the samples treated with buffer or with endolysin. This assay was repeated three times and the error bars indicate the standard deviations.
- Target Site Identification
Crude peptidoglycan of E. coli DH5α was extracted as previously described with slight modifications [7 , 20] . The crude cell wall was harvested from disrupted E. coli by centrifugation (27,000 × g , 5 min). It was resuspended in 4% sodium dodecyl sulfate solution and boiled for 10min. After several washings, the crude peptidoglycan solution (160 μl) was suspended in 50 mM Tris-HCl (pH 8.0) mixed with the SPN9CC endolysin (5 μg), and the change in optical density at 600 nm was measured using a Sunrise microplate absorbance reader (Tecan, Switzerland). To more accurately investigate the SPN9CC endolysin cleavage site, the peptidoglycan hydrolysate supernatant was subjected to the following protocol. To confirm glycosidase activity, the amount of the reducing sugar was measured using a modification of a previously reported method [33] . Peptidoglycan hydrolysates were mixed with an equal volume of 0.05% (w/v) aqueous potassium ferricyanide and 0.53% (w/v) sodium carbonate/0.065% (w/v) potassium cyanide. After boiling for 15 min, a ferric ion reagent (final 0.15% (w/v) ferric ammonium sulfate/0.1% (w/v) SDS in 0.025 M sulfuric acid) was added, and the OD 690 was measured. To confirm the amidase activity, the peptidoglycan hydrolysate was incubated with 1.0 M NaOH for 30 min. After the addition of 0.5 ml of 0.5 M H 2 SO 4 and 5 ml of concentrated H 2 SO 4 , the mixture was boiled f or 5 min. After cooling, 0.05 ml CuSO 4 a nd 0.1ml PHD solutions (1.5% p -hydroxyphenyl in 96% ethanol) were added. After incubation for 30 min, the OD 560 was measured [10 , 12] . Before determining the peptidase activity, the peptidoglycan was acetylated using half-saturated sodium acetate and 10 mM acetic anhydride [27] . To determine the peptidase activity, 4% NaHCO 3 and 0.1% trinitrobenzene sulfonic acid solutions (final concentrations) were added to the acetylated peptidoglycan. After acidification using 1N HCl for 1 h, the OD 340 was measured. In all target identification assays, a peptidoglycan suspension that had not been treated with the SPN9CC endolysin and the same amount of the endolysin solutions were used as negative controls. The standard deviations of four independent experiments were represented by error bars.
Results and Discussion
- Expression and Purification of SPN9CC Endolysin
The genome of the SPN9CC bacteriophage contains a lysis cluster that includes genes that encode holin (SPN9CC_0042), endolysin (SPN9CC_0043), and Rz/Rz1-like proteins (SPN9CC_0044) [39] . To verify the lytic activity of the endolysin, a recombinant plasmid was constructed. The endolysin coding region was cloned into pET29b to express recombinant SPN9CC endolysin with a histidine tag at its C-terminal region. The overexpression of the recombinant endolysin was induced by the addition of a final concentration of 1 mM of IPTG to E. coli harboring the recombinant plasmid. The SPN9CC endolysin was purified using Ni-NTA affinity chromatography, and the recombinant protein was detected at the expected size (approximately 17 kDa) by SDS-PAGE ( Fig. 1 A).
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Purification of recombinant SPN9CC endolysin. (A) E. coli cell cultures harboring the backbone plasmid (lane 1) or recombinant plasmid (lane 2) were induced with 1 mM IPTG, and the total cell lysates were separated on a 15% acrylamide gel. The recombinant SPN9CC endolysin (~17 kDa) was purified using a Ni-NTA agarose resin (lane 3). M, molecular weight markers. (B) The lytic activity of the purified SPN9CC endolysin was confirmed using EDTA-treated E. coli cells as a substrate. Different concentrations of endolysin were added to the substrate at time zero.
- Lysis activity of SPN9CC Endolysin
E. coli cells that had been treated with EDTA to enhance the permeability of their outer membranes were used as substrate for the purified endolysin, because the SPN9CC endolysin showed the highest activity against E. coli (see below). The lytic activity of the SPN9CC endolysin was assayed by measuring the optical density of the endolysintreated substrate suspensions. The addition of 0.1 μg/ml endolysin resulted in increased lysis compared with buffer alone, and the enzymatic activity increased as the endolysin concentration was increased up to 5 μg/ml ( Fig. 1 B). The lytic activity of the SPN9CC endolysin was strong and fast, and the lysis activity was similar to that of commercial lysozyme purified from chicken egg whites [20] .
- Optimal pH and Temperature Conditions for Enzymatic Activity
The SPN9CC endolysin was active over a broad range of temperatures (24℃ to 65℃). It showed similar activities at neutral and basic pH values (pH 7.0 to 9.5) but showed maximal activity at pH values of 7.5 to 8.5 and at 50℃ ( Fig. 2 ).
To verify the thermal stability of the SPN9CC endolysin, endolysin aliquots were incubated at different temperatures (37℃, 45℃, 55℃, or 65℃) for 2 h. Thermal treatment at temperatures up to 45℃ for 30 min or at 37℃ for 2 h did not significantly inhibit enzyme function; however, elevated temperatures (55℃ or 65℃) or longer incubation times (1 or 2 h) had a negative effect on the activity (data not shown).
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Optimum conditions for the enzymatic activity of the SPN9CC endolysin. The antimicrobial assay was performed at different temperatures for 3 min in 50 mM Tris-Cl buffer, pH 8.0 (A), and in buffers with different pH values for 10 min at room temperature (B). The lytic ability was detected by measuring the optical density of the enzymetreated substrate (EDTA-treated E. coli), and the relative lysis was calculated in proportion to the highest amount of lysis obtained.
- Antimicrobial Spectrum of SPN9CC Endolysin
Eight gram-positive and 23 gram-negative strains were assessed for their susceptibility to SPN9CC endolysin ( Fig. 3 ). All of the tested gram-negative strains were lysed by the SPN9CC endolysin (0.5 μg/ml) within 5 min. The highest activities were observed against E. coli strains JM109 and MG1655 and Pseudomonas putida KCTC 1643. However, no lysis was detected in gram-positive strains. Thus, it was concluded that the SPN9CC endolysin had a wide spectrum of antimicrobial activity against gramnegative bacteria.
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The antimicrobial spectrum of the SPN9CC endolysin. After the endolysin was added to each suspension of gram-negative or gram-positive bacteria, the optical densities were measured. The relative lysis activity refers to the optical density in comparison with the optical density prior to the enzymatic reaction. For the gram-negative strains, the EDTA treatment was performed before the addition of the endolysin.
- Target Site Identification
Bioinformatic analysis indicated that the SPN9CC endolysin possessed lytic transglycosylase activity. Lytic transglycosylases cleave the β-1,4 glycosidic bond between N -acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc), and they are widely distributed among gram-negative bacteria. In contrast to lysozymes that act on the same glycosidic linkage, lytic transglycosylases do not hydrolyze glycosidic bonds but rather produce 1,6-anhydromuramoyl residues [14 , 37] .
E. coli peptidoglycan was used as a substrate to confirm the predicted target site. The SPN9CC endolysin was able to degrade E. coli peptidoglycan, and the amount of reducing sugar was significantly increased as the peptidoglycan was degraded ( Figs. 4 A and 4 B). These results showed that the SPN9CC endolysin possessed glycosidase activity. The buffer and enzyme used in this experiment did not affect the results. To examine whether the SPN9CC endolysin might have other target sites, amidase activity was also analyzed using the same enzyme; however, no difference was found between the endolysin lysate and the peptidoglycan suspension ( Fig. 4 C). To analyze the peptidase activity, the peptidoglycan was acetylated with acetic anhydride to remove preexisting amine residues. It was difficult to observe the hydrolysis of the acetylated peptidoglycan by the SPN9CC endolysin by measuring the optical density of the peptidoglycan suspension, because the lytic activity was decreased against acetylated peptidoglycan; however, hydrolysis could be confirmed by detecting the reducing sugar. As shown in Fig. 4 D, the SPN9CC endolysin did not have peptidase activity. Thus, we concluded that the SPN9CC endolysin cleaves only the glycosidic bond of peptidoglycan.
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Target site of the SPN9CC endolysin. (A) Peptidoglycan was extracted from E. coli DH5α cells and mixed with the SPN9CC endolysin. After a 1 h reaction at room temperature, the optical density (600 nm) was measured. The glycosidase activity (B), amidase activity (C), and peptidase activity (D) of the peptidoglycan hydrolysate digested with the SPN9CC endolysin were analyzed.
- Exogenous Lytic Activity of SPN9CC Endolysin Against Intact Gram-Negative Bacteria
It was interesting to note that the lytic activity of the SPN9CC endolysin against E. coli without EDTA pretreatment could be observed when the amount of endolysin and incubation time were increased. After 1 h reaction, the SPN9CC endolysin (300 μg/ml) reduced the number of viable E. coli cells by approximately 2 logs, but the addition of EDTA did not increase the SPN9CC endolysin activity ( Fig. 5 A). When the incubation time was increased to 2h, the addition of EDTA enhanced the lytic activity of the SPN9CC endolysin against E. coli . A combination of 300 μg/ml endolysin and 1 mM EDTA resulted in a reduction in CFUs by approximately 4 logs; this activity was approximately 2-fold higher than the SPN9CC endolysin treatment without EDTA ( Fig. 5 B). However the treatments of EDTA (1 or 5 mM) alone did not significantly affect cell viability, suggesting that the addition of EDTA probably increases the outer membrane permeability, helping the lytic action of SPN9CC endolysin. There are few reports of endolysins acting on intact gram-negative bacteria because endolysins cannot penetrate the outer membrane to reach the peptidoglycan layer of gram-negative bacteria. To elucidate the mechanism of SPN9CC endolysin effective against gramnegative bacteria without outer membrane permeabilization, the amino acid sequence of SPN9CC endolysin was compared with other endolysins.
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Lytic activities of the purified SPN9CC endolysin. The viable cell numbers (CFUs) of E. coli after the addition of 100 or 300 μg/ml of SPN9CC endolysin were examined. Different concentrations of EDTA (0, 1, or 5 mM) were added to the cells at the same time that the endolysin was added. The enzymatic reactions were performed for 1 h (A) or 2 h (B).
- N-Terminal Hydrophobic Domain of SPN9CC Endolysin
Generally, endolysins cannot easily access the peptidoglycan of gram-negative bacteria, because the outer membrane prevents entrance to the interior of the bacteria. This is one obstacle to be overcome before bacteriophage endolysins can be used as an antibiotic substitute against gramnegative bacteria [13 , 19] . However, two endolysins are capable of lysing bacterial cells without any pretreatment. Morita et al . [29] demonstrated the exogenous antibacterial action of the B. amyloliquefaciens phage endolysin against P. aeruginosa PAO1. Similarly, LysAB2 produced from an Acinetobacter baumannii bacteriophage exhibited bacteriolytic activity against a number of gram-negative and grampositive bacteria when applied exogenously [19] . In both endolysins, the C-terminal hydrophobic regions possess putative amphipathic helix domains that may allow the endolysins to pass through the outer membrane. They speculated that the C-terminal regions of the endolysin interact with or penetrate the cell envelope; subsequently, the N-terminus of the endolysin, which may harbor the catalytic domain, approaches the peptidoglycan layer, causing the lysis of the bacterial cell. Similar to these reported endolysins, the SPN9CC endolysin showed exogenous antibacterial activity without outer membrane permeabilization.
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Hydropathy plot and functional domain analysis of the SPN9CC endolysin (A) and the N-terminal sequences of the SPN9CC endolysin, R21, and LyzP1 (B). (B) White boxes indicate the N-terminal signal-anchor-release (SAR) domains, and the black boxes indicate the catalytic triads (Glu-8aa-Asp/Cys-5aa-Thr).
The functional protein domain analysis of the SPN9CC endolysin showed that it contains a family 24-glycoside hydrolase catalytic domain (PF00959) in its C-terminal region. Furthermore, a transmembrane helix was detected in the N-terminal region of the SPN9CC endolysin ( Fig. 6 A), and it could be possible that this region, the terminal signal-anchor-release (SAR) domain, is responsible for outer membrane penetration. This is a similar domain structure to that of previously reported Lyz P1 and R 21 lysozymes containing N-terminal SAR domains. The catalytic triad (Glu-8aa-Asp/Cys-5aa-Thr) of these lysozymes was also identified in the SPN9CC endolysin between amino acids 37 and 5 2 ( Fig. 6 B) [41 , 45] . Previous studies of the Lyz P1 and R 21 lysozymes showed that the N-terminal SAR domain is associated not only with endolysin exportation from the cytoplasm to the periplasm but also with the regulation of its lytic activity [41 , 45] . To test the functions of the SAR domain in the SPN9CC endolysin, N-terminal truncated SPN9CC endolysins with deletions from amino acids 2 to 16, 2 to 17, or 2 to 18 were constructed. These Nterminal truncated endolysins were purified and tested for lytic activity against E. coli ; however, none of the truncated endolysins showed lytic activity against E. coli regardless of EDTA treatment (data not shown). Thus, the SAR domain of the SPN9CC endolysin may be involved in the lytic activity of the endolysin. However, the truncated endolysins may be misfolded, so further studies regarding the SAR domain functions of the SPN9CC endolysin should be performed in the future.
Acknowledgements
This work was carried out with the support of the the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009842)”, Rural Development Administration, Republic of Korea.
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