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Di¡erent contributions of the outer and inner R-core residues of lipopolysaccharide to the recognition by spike H and G proteins of bacteriophage PX174 Minoru Inagaki , Tomoko Kawaura, Hirohito Wakashima, Muneharu Kato, Shiro Nishikawa, Naoki Kashimura Department of Life Science, Faculty of Bioresources, Mie University, 1515 Kamihama, Tsu, Mie 514-8507, Japan Received 25 February 2003; received in revised form 18 June 2003 ; accepted 28 June 2003 First published online 23 August 2003
Abstract The binding of spike H and G proteins of bacteriophage PX174 with lipopolysaccharides (LPSs) were evaluated by a competitive enzyme-linked plate assay using the biotin-labeled LPS of Escherichia coli C, one of a host strain, and the non-labeled LPSs having different R-core polysaccharide lengths. H protein promptly decreased its affinity when some saccharide residues were truncated from the outer R-core. However, G protein showed significant affinity to the LPSs lacking all the residues of the outer R-core and some of the inner R-core. Thus, G protein rather than H protein well recognized the residues of the inner R-core of LPS. 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Bacteriophage PX174; Host recognition; Lipopolysaccharide ; Receptor; Spike protein
1. Introduction Bacteriophage PX174 is a small icosahedral virus of 26 nm diameter, and consists of a single-stranded circular DNA and four capsid proteins, F, G, H and J [1]. On each twelve vertices of an icosahedron, PX174 has spikes that are constituted of an H and ¢ve G proteins [2]. An electron microscopic study showed that PX174 adsorbed itself on the surface of host bacterial cell by one of the spikes [3]. PX174 was also reported to recognize lipopolysaccharide (LPS) as a receptor on the surface of host enterobacteria such as Escherichia coli C, Salmonella enterica serovar Typhimurium TV119 (S. Typhimurium TV119), Shigella sonnei phase II, etc. [4]. The spike proteins, hence,
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[email protected] (M. Inagaki).
have been believed to recognize LPS. However, there was no direct evidence for the binding of spike H and/or G proteins with LPS. In our previous studies, we prepared the fused H proteins with maltose-binding protein (MBPH) [5] or histidine tag (HisH) [6,7], and demonstrated their speci¢c binding to the LPSs of the host strains of PX174. In addition, the fused G protein with histidine tag (HisG) was also found to bind to the LPSs [8]. The spike H and G proteins have been estimated to play quite di¡erent roles in the infection process of PX174: G protein provides a channel for the injection of phage DNA into host cell [9], and H protein penetrates through the host membrane along with the DNA [10]. For this reason, the way of recognition toward LPS would be di¡erent from each other, and we thus intended to compare the spike H and G proteins in relation to their function. In this report, the contributions of the outer and inner R-core polysaccharide residues of LPS for the recognition by the spike proteins were quantitatively evaluated by a competitive enzymelinked plate assay between the biotin-labeled LPS of (B-LPS) E. coli C, one of the native host strains, with the non-labeled LPSs of the host and non-host strains that have various R-core polysaccharide sequences and lengths.
0378-1097 / 03 / $22.00 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(03)00601-3
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2. Materials and methods 2.1. Materials The LPS of E. coli C strain was extracted from the cultured cells [11] by the phenol-chloroform-petroleum ether extraction method [12]. The LPSs of S. Typhimurium wild-type, TV119 and SL684 strains, and the E. coli
EH100, J5, F583, and O111:B4 strains were purchased from Sigma (St. Louis, MO, USA). The streptavidin-peroxidase conjugate (STP-POD) was purchased from Zymet (South San Francisco, CA, USA). An assay plate (Immuno Plate II) was a product of Nalge Nunc International (Tokyo, Japan). Unless otherwise speci¢ed, all chemicals were the guaranteed grade reagents of Nacalai Tesque (Kyoto, Japan).
Fig. 1. Chemical structures of LPSs. The structures of the LPSs used in this study are summarized with references to the recent reports [4,16^20]. Some chemical linkages with question marks are not fully identi¢ed, and dotted lines in the ¢gure indicate non-stoichiometric substitutions.
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2.2. Sodium deoxycholate^polyacrylamide gel electrophoresis (DOC^PAGE) DOC^PAGE analysis was performed according to the reported procedure [13] with some modi¢cations. The running gel consisted of 15% acrylamide, 0.4% bis-acrylamide, and 0.9% DOC in 375 mM Tris^HCl (pH 8.8). The stacking gel was made of 3% acrylamide, 0.08% bis-acrylamide, and 0.5% DOC in 125 mM Tris^HCl (pH 6.8). LPSs were dissolved in a sample bu¡er containing 0.5% DOC and 20% glycerol in 80 mM Tris^HCl (pH 6.8). The electrode bu¡er contained 0.2% DOC, 384 mM glycine, and 50 mM Tris (pH 8.5). After pre-electrophoresis at 25 mA for 90 min, LPSs in the sample bu¡er were charged onto the gel and developed at 10 mA using a fresh electrode bu¡er. The developed gel was washed with 10% acetic acid in 40% aqueous methanol to remove DOC, and stained by the Silver Stain II Kit (Wako Pure Chemical Industries, Osaka, Japan) after treatment with 1% periodic acid and 3% acetic acid in 40% aqueous methanol for 1.5 h at 25‡C for sensitization [14]. 2.3. Fused spike H and G proteins of bacteriophage PX174 The fused spike proteins with histidine tag (HisH and HisG) were expressed in E. coli harboring the expression vectors constructed from PX174 RF DNA (Toyobo, Osaka, Japan) and pQE-30 (Qiagen, Hilden, Germany), and puri¢ed by two-step chromatography using Ni-NTA agarose (Qiagen) and DEAE or CM-cellulo¢ne ion exchanger (Seikagaku Corp., Tokyo, Japan) according to our previous reports [6^8]. Protein concentrations were measured by the Coomassie Brilliant Blue dye-binding method (Protein Assay Kit, Bio-Rad, Hercules, CA, USA), using bovine serum albumin (BSA) as a standard.
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ml31 ) and 30% H2 O2 (1 Wl ml31 ) in 50 mM sodium citrate-phosphate bu¡er (pH 5.0)] was dropped at room temperature. The peroxidase reaction was allowed for 5 min and stopped by addition of 50 Wl of 3 M HCl. Absorbance of each well was measured at 490 nm with reference to the absorbance at 630 nm by using an Immuno Mini NJ-2300 plate reader (Nalge Nunc International).
3. Results 3.1. Chemical structures and DOC^PAGE analysis of LPSs LPS consists of three distinct regions: lipid A, R-core polysaccharide, and O-repeating unit [15]. The structures of LPSs used in this study are shown in Fig. 1. Some chemical linkages have not been fully determined, thus, the most reasonable structures are summarized with reference to the recent structural studies [4,16^20]. The LPSs were analyzed by DOC^PAGE and the developed gel was visualized by silver staining (Fig. 2). The LPSs of the PX174-insensitive strains, S. Typhimurium wild-type and E. coli O111:B4 have O-repeating units in addition to the R-core [16] and showed characteristic ladder-like patterns on the DOC^PAGE. The host strains of PX174 are limited to the Ra strains that have complete R-core sequences on their LPSs and have no O-repeating unit in addition to the R-core. However, some types of R-cores such as the E. coli R1-R4, Salmonella, and Shigella cores are acceptable as the receptors [4]. The LPS of E. coli C, one of the native host strains of PX174, is made of a complete the E. coli
2.4. Competitive enzyme-linked plate assay HisH (10 Wg ml31 ) or HisG (4 Wg ml31 ) in 100 Wl of Tris bu¡er with 100 mM NaCl (pH 7.4) (TBS) was adsorbed onto the wells in an assay plate (Immuno Plate II) at 4‡C for 3 h. After being rinsed three times with 150 Wl of TBS (50 mM Tris^HCl with 100 mM NaCl, pH 7.4), the wells were coated with 150 Wl of 0.5% BSA in TBS at 4‡C for 4 h, and then rinsed three times with 200 Wl of a wash bu¡er (0.05% BSA and 0.05% Tween 20 in TBS) and three times with 200 Wl of TBS. The B-LPS of E. coli C (10 Wg ml31 ) in 50 Wl of TBS and the unlabeled LPSs (0^500 Wg ml31 ) in 50 Wl of TBS were added to the wells, and the plate was kept at 4‡C overnight. After the wells were rinsed six times with the wash bu¡er, STP-POD in the wash bu¡er (100 Wl) was added for coupling with the B-LPS bound on the plate for 3 h at room temperature. After removal of excess STP-POD by four times washing with the wash bu¡er (200 Wl), 100 Wl of a substrate solution [o-phenylenediamine dihydrochloride (OPD) (2 mg
Fig. 2. Comparison of LPSs by silver-stained DOC^PAGE. LPSs of various strains were developed by DOC^PAGE and visualized by silver staining. Lane 1: the LPS of S. Typhimurium wild-type (smooth, 12 Wg), lane 2: E. coli O111:B4 (smooth, 10 Wg), lane 3: E. coli C (Ra, 0.4 Wg), lane 4: S. Typhimurium TV119 (Ra, 0.4 Wg), lane 5: E. coli EH100 (Ra, 0.4 Wg), lane 6: S. Typhimurium SL684 (Rc, 0.4 Wg), lane 7: E. coli J5 (RcPþ , 0.26 Wg), and lane 8: E. coli F583 (Rd2 , 0.26 Wg ml31 ). The experimental details are explained in Section 2.
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3.2. Quantitative evaluation of the a⁄nities of the LPSs having various R-core sequences and lengths to the spike H and G proteins
Fig. 3. Enzyme-linked competitive assay using the B-LPS of E. coli C and the non-labeled LPSs of various strains for the binding with HisH (A) and HisG (B). The binding of the B-LPS of E. coli C (5 Wg ml31 ) with HisH or HisG was inhibited by the non-labeled LPSs having various polysaccharide sequences (0^500 Wg ml31 ): the LPSs of E. coli C (Ra) (open squares), S. Typhimurium TV119 (Ra) (closed circles), E. coli EH100 (open reversed triangles), S. Typhimurium SL684 (Rc) (open circles), E. coli J5 (RcPþ ) (closed squares), E. coli F583 (Rd2 ) (closed triangles), and E. coli O111 :B4 (open triangles). The solid lines are the theoretical curves drawn according to Eq. 1, using the calculated I50% values in Table 1. The experimental details are described in Section 2.
R1-type core [4,17,18] and showed a simple band on the DOC^PAGE. The E. coli R1 core consists of two galactose (Gal), three glucose (Glc), three L-glycero-D-mannoheptose (Hep), and two 3-deoxy-D-manno-octulosonic acid (KDO) residues. S. Typhimurium TV119 (Ra) and E. coli EH100 (Ra) have complete Salmonella-type and E. coli R2-type cores [17,18], and they are PX174-sensitive strains [4]. These Ra-type LPSs showed the same mobility on the DOC^PAGE. The LPSs of the PX174-insensitive strains, S. Typhimurium SL684 (Rc) [17], E. coli J5 (RcPþ ) [19], and E. coli F583 (Rd2 ) [20], have partial structures of the Salmonella, E. coli R3, and E. coli R1type cores, respectively. These LPSs were used in the following enzyme-linked assay.
In our previous study, we established the enzyme-linked plate assay for detection of the interaction by spike proteins with the B-LPSs of PX174-sensitive and -insensitive strains [6^8]. Only the labeled LPS is detectable in the assay by means of coupling with STP-POD, which has an activity for oxidation of OPD ; consequently, the interaction of the non-labeled LPS with the spike proteins is detectable as inhibition of the binding of the B-LPS. The relative binding a⁄nities of the unlabeled LPSs having various polysaccharide sequences and lengths were thereby evaluated by the competitive binding with the B-LPS of E. coli C, one of the LPSs of native host strains (Fig. 3). HisH or HisG in a ¢xed concentration was adsorbed onto assay plates, and subjected to the interaction with a series of mixtures of the B-LPS of E. coli C (5 Wg ml31 ) and the non-labeled LPSs (0^500 Wg ml31 ). The absorbance at 490 nm at the wells without the non-labeled LPSs was de¢ned as 100%, and the reduced absorbance caused by inhibition was plotted in percentages (%) against the initial concentrations of the non-labeled LPSs, [LPS]0 (Wg ml31 ). BSA, which was reported to bind with a wide variety of hydrophobic compounds including LPS [21], was used as a coating agent for hydrophobic polystyrene surface of the assay plate. However, the background binding of B-LPS to BSA was suppressed to a negligible level by insistently washing the plate with a bu¡er containing BSA and a non-ionic surfactant, Tween 20. The binding of the B-LPS of E. coli C to HisH and HisG was completely inhibited by the non-labeled LPS of E. coli C, and descending saturation curves were observed for both cases. The inhibition was generally strong by the LPSs of the PX174-sensitive strains and weak by those of the insensitive strains. Since the magnitude of inhibition was di¡erent for di¡erent kinds of LPSs and spike proteins, we introduced Eq. 1 for quantitative estimation of the a⁄nity of various LPSs, where Imax (%) is the maximum inhibition, I50% (Wg ml31 ) is the concentration required for a 50% inhibition, and [LPS]0 (Wg ml31 ) is the initial concentration of non-labeled LPS. The equation corresponds to the subtraction of the binding of the nonlabeled LPSs from the maximum binding (100%) of the labeled LPS, and Imax and I50% are functionally similar to the maximum velocity, Vmax , and inhibition constant, Ki , in the conventional enzyme kinetics: Relative binding ¼ 1003
I max U½LPS0 I 50% þ ½LPS0
ð1Þ
Since the mode of inhibition by the non-labeled LPSs toward the labeled LPS was regarded as competitive in this case, the values of Imax were expected to be 100%. Indeed, the Imax for all LPSs were converged to nearly
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100% with some deviations when I50% as well as Imax was optimized by the least-squares ¢tting of the observed points to Eq. 1. Such deviations tended to increase for the LPSs of low a⁄nity; however, there was no reason to substitute the Imax value other than 100%. Thus, the Imax value was ¢xed at 100%, and the I50% values for all kinds of LPSs were calculated by the least-squares ¢tting to Eq. 1 (Table 1). The solid lines in Fig. 3 are the theoretical curves drawn according to Eq. 1 using the calculated values. The absolute values of I50% were variable for every experiment; however, their relative magnitudes were reproducible. The binding a⁄nity of each LPS was quantitatively evaluated by dividing the I50% value of the LPS of E. coli C by those of the LPSs. This is the devisable point for extracting absolute a⁄nity data from a kind of semiquantitative result by the enzyme-linked plate assay. Based on the a⁄nity of LPS of E. coli C as 100%, the a⁄nity of the LPS of S. Typhimurium TV119 (Ra) for HisH was almost comparable (110 S 8%); however, that for HisG was inferior (72 S 7%) to the LPS of E. coli C. The LPS of E. coli EH100 showed almost the same a⁄nity for HisG (72 S 3%) as the LPS of S. Typhimurium TV119. These two LPSs have very similar structures in the R-core: only the third residues from the non-reducing end are di¡erent from each other (see Fig. 1). The LPSs of S. Typhimurium SL684 (Rc) and E. coli J5 (RcPþ ) lacking four of ¢ve hexose residues of the Salmonella and E. coli R3-type cores showed limited a⁄nities for both HisH and HisG. Their a⁄nities were decreased signi¢cantly for HisH rather than HisG. A decreased a⁄nity was also observed for HisH than HisG for the LPS of E. coli F583 (Rd2 ) lacking all hexose and two or three heptose residues from the E. coli R1-type core. The least a⁄nity was observed for a smooth LPS of E. coli O111:B4 for both HisH and HisG. This observation is well consistent with the fact that PX174 cannot infect any smooth strains that have O-repeating units in addition to the R-core [4]. The LPS binding by spike H protein was determined predominantly by the hexose residues of the outer R-core and only partly by
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Fig. 4. SDS^PAGE and native-PAGE analysis of HisG. A: SDS^PAGE (12.5%); lane 1, markers (Nacalai Tesque) containing myosin (200), Lgalactosidase (116), BSA (66), ovalbumin (45), carbonic anhydrase (31), trypsin inhibitor (22), lysozyme (14), and aprotinin (6.5 kDa); lane 2, HisG heated with 1% SDS at 100‡C for 5 min; lane 3, HisG not heated. B: Native-PAGE (10%); lane 4, BSA (66 kDa) as a typical example showed a weak band of dimer in addition to a monomer; lane 5, HisG in native conditions showed one major and two minor bands of oligomers. This HisG preparation showed a slightly deformed peak around 45 kDa by gel ¢ltration, indicating that a main band of oligomer on the native-PAGE gel corresponds to a dimer of HisG.
the heptose residues of the inner R-core, while the contributions of heptose and KDO residues of the inner R-core are more signi¢cant for spike G protein than those for H protein. Jazwinski et al. [22] reported about 60% of binding e⁄ciency of the PX174 particle to the S. Typhimurium SH180 (Ra) cell compared with the E. coli C cell. Because the SH180 strain has the same R-core structure as the TV119 strain, the present data, which showed 72% of af¢nity to HisG for the LPS of S. Typhimurium TV119 compared to that for E. coli C, are very well consist with their result. Thus, the binding selectivity of the spike G protein to the LPS of host cell well explains the binding selectivity of a whole PX174 particle to the host cell.
Table 1 Relative binding a⁄nities of the LPSs of various strains to HisH and HisG Origin of LPS
E. S. E. S. E. E. E. a
coli C Typhimurium TV119 coli EH100 Typhimurium SL684 coli J5 coli F583 coli O111:B4
The from b The c Not
Chemotype
Ra Ra Ra Rc RcPþ Rd2 smooth
HisH
HisG
I50% a (Wg ml31 )
A⁄nityb (%)
I50% a (Wg ml31 )
A⁄nityb (%)
5.2 S 0.3 4.7 S 0.3 ^c 37.2 S 4.1 163 S 37 365 S 50 2821 S 210
100 S 0 110 S 8 ^c 14 S 2 3.2 S 0.7 1.4 S 0.2 0.18 S 0.01
16.6 S 0.8 23.0 S 2.1 23.0 S 1.0 85.1 S 9.6 ^c 47.8 S 9.7 884 S 50
100 S 0 72 S 7 72 S 3 19 S 2 ^c 35 S 7 1.9 S 0.1
values of I50% were calculated by the non-linear least-squares ¢tting of the data points of Fig. 3 to Eq. 1. Standard deviations were calculated more than three independent experiments, and are shown as S values. relative a⁄nities were calculated as percentages by comparing the magnitudes of I50% values of various LPSs with that of the LPS of E. coli C. determined.
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4. Discussion In the PX174 particle, the spike G protein forms pentameric spike at the twelve vertices of an icosahedral capsid [2,3]. The G protein was reported to assemble by itself without a help of sca¡olding or chaperone proteins during morphogenesis in the infected cell [1,9]. The sodium dodecyl sulfate (SDS)^PAGE of HisG showed oligomer formation in spite of the presence of 1% SDS (Fig. 4A). The monomer as well as the dimer of HisG was detected at 23.2 and 46.5 kDa, respectively (lane 2). The ratio of dimer to monomer was apparently increased when a heating treatment in an SDS sample bu¡er was omitted (lane 3). The native-PAGE also showed a major and two minor bands of oligomers (lane 5). The averaged molecular mass of this HisG preparation was calculated to be ca. 45 kDa by gel ¢ltration using a cellulo¢ne GCL-1000m column (1.2 cmPU250 cm) in comparison with the proteins for gel ¢ltration standard (Bio-Rad). Thus, HisG used in the present study was thought to exist mainly as dimer, partially as trimer, and few as monomer in solution. The spike G protein in the virion was shown to fold in eightstranded anti-parallel L-barrel [9]. The N-terminal eight residues and the C-terminal 10 residues of amino acids are stretched out from the L-barrel to interact with an adjacent G protein. Since HisG has an extra sequence of 18 amino acid residues including hexa histidine tag on the N-terminal, the pentamer formation is thought to be inhibited. The pentamer formation would be not essential for the LPS binding, and there is at least an LPS binding site on each G protein. In the infection process of PX174, one of 12 molecules of spike H protein in the virion was reported to penetrate through the lipophilic membrane of bacterial cell along with the phage DNA [10]. On the other hand, the spike G and the capsid F proteins were found to remain outside of the cell as a vacant phage shell after the infection process [23]. The present data showed that HisH has no practical selectivity between two Ra-type LPSs of E. coli C and S. Typhimurium TV119, while the saccharide sequences are di¡erent each other. Thus, there is a possibility that H protein recognizes a balance of hydrophobic and hydrophilic residues rather than the R-core sequence of LPS. In the previous study, we measured the dissociation constants, Kd , of HisH and HisG to the LPS of E. coli C by £uorometric titration, based on the dose-dependent £uorescence change of the proteins in the presence of the LPS. The Kd values were 7.0 WM for HisH [7], and 0.16 WM for HisG [24], respectively. The a⁄nity to HisG is consequently 44-fold stronger than that to HisH. When the absolute scale of Kd was adapted to the present order of a⁄nities of LPSs in Table 1, the a⁄nity of the secondworst recognized LPS of S. Typhimurium SL684 (Rc) to HisG is still stronger than that of one of the best-recognized LPS of E. coli C (Ra) to HisH. It is likely in the infection process, PX174 recognizes the receptor LPS of
bacterial cell roughly by H protein and then strictly by G protein in the spike device. Some host range mutants of PX174 were isolated and characterized as mutations in the gene G spike protein [25]. Although a host range mutant was also found in the gene F capsid proteins [26], mutations in the gene F rather concern with the rate of infection such as temperature sensitivity for DNA ejection reaction [27,28]. Moreover, the gene G mutants having an increased number of positively charged amino acid residues showed an increased adsorption velocity to the host cell [27,29]. This indicates that the spike G protein has a role of adsorption of the phage particle to the bacterial cell, and is consistent with the present observation that HisG rather than HisH showed stricter recognition to the inner R-core of the LPSs including negatively charged phosphate and KDO residues. The process of PX174 for attachment and penetration to the host bacterial cell was a well-known two-step process: reversible attachment and irreversible adsorption followed by DNA penetration [1,23,29]. A glucose molecule bound on the depression of capsid F protein near the three-fold axis of an icosahedron was found by X-ray crystallographic study of PX174 [30]. The glucose was thought to be a fragment of LPS of the E. coli cell and was co-puri¢ed with the phage particle. Amino acid sequences around the glucose-binding site are completely conserved among all the microviridae family of phages including PX174, G4, K3, and PK [9,30]. The site is hence thought to relate the initial reversible step of attachment of PX174 to the host cell for infection. After the reversible step, the recognition by spike G as well as H proteins to the LPS would result in the irreversible adsorption of PX174. This phenomenon is most likely analogous to the interaction of the ¢bers of tailed phages with the LPS of host bacterial cell. The longtail ¢bers ¢rst recognize the LPS and are responsible for the reversible attachment of virion to the host cell [31]. And then the short-tail ¢bers bind irreversibly to the LPS, which serves as a trigger for contraction of the tail sheath for penetration of the cell envelop by the tail tube [32]. It is impressive that such a two-step adsorption process of phages to the host cell is commonly performed between tailed and icosahedral phages using di¡erent kinds of devices. Taking into account a functional resemblance among the devices of both types of phages, the events that are needed to happen in the infection process must be consistent. The change in the shape of the base plate of the tailed phages from hexagonal into six-pointed star triggering the contraction of tail sheath for DNA injection is in correspondence with that the conformation change of spike H and G proteins in case of the binding with receptor LPS may result in a transition of capsid conformation for DNA penetration in the icosahedral phages. The di¡erent contributions of R-core saccharide residues in the LPS recognition between the spike H and G proteins of the icosahedral phages are also in correspondence with that di¡erent saccharide residues of the LPS
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may be recognized by the short- and long-tail ¢bers of the tailed phages.
Acknowledgements One of the authors (T.K.) gratefully acknowledges the support of the Research Fellowship for Young Scientists sponsored by the Japan Society for the Promotion of Science. The authors thank the reviewer and the editor for their helpful comments to improve the manuscript.
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