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JOURNAL OF BACTERIOLOGY, May 1995, p. 26842694 0021-9193/95/$04.

00 0 Copyright 1995, American Society for Microbiology

Vol. 177, No. 10

Physical and Functional S-Layer Reconstitution in Aeromonas salmonicida


RAFAEL A. GARDUNO, BARRY M. PHIPPS,
AND

WILLIAM W. KAY*

Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Victoria, British Columbia V8W 3P6, Canada
Received 9 November 1994/Accepted 6 March 1995

The various functions attributed to the S-layer of Aeromonas salmonicida have been previously identied by their conspicuous absence in S-layer-defective mutants. As a different approach to establish the multifunctional nature of this S-layer, we established methods for reconstitution of the S-layer of A. salmonicida. Then we investigated the functional competence of the reconstituted S-layer. S-layers were reconstituted in different systems: on inert membranes or immobilized lipopolysaccharide (LPS) from puried S-layer protein (Aprotein) or on viable cells from either A-protein or preassembled S-layer sheets. In the absence of divalent cations and LPS, puried A-protein in solution spontaneously assembled into tetrameric oligomers and, upon concentration by ultraltration, into macroscopic, semicrystalline sheets formed by oligomers loosely organized in a tetragonal arrangement. In the presence of Ca2 , puried A-protein assembled into normal tetragonal arrays of interlocked subunits. A-protein bound with high afnity (Kd, 1.55 10 7 M) and specicity to high-molecular-weight LPS from A. salmonicida but not to the LPSs of several other bacterial species. In vivo, A-protein could be reconstituted only on A. salmonicida cells which contained LPS, and Ca2 affected both a regular tetragonal organization of the reattached A-protein and an enhanced reattachment of the A-protein to the cell surface. The reconstitution of preformed S-layer sheets (produced by an S-layersecreting mutant) to an S-layer-negative mutant occurred consistently and efciently when the two mutant strains were cocultured on calcium-replete solid media. Reattached A-protein (exposed on the surface of S-layer-negative mutants) was able to bind porphyrins and an S-layer-specic phage but largely lacked regular organization, as judged by its inability to bind immunoglobulins. Reattached S-layer sheets were regularly organized and imparted the properties of porphyrin binding, hydrophobicity, autoaggregation, adherence to and invasion of sh macrophages and epithelial cells, and resistance to macrophage cytotoxicity. However, cells with reconstituted S-layers were still sensitive to complement and insensitive to the antibiotics streptonigrin and chloramphenicol, indicating incomplete functional reconstitution. S-layers cover the cell surfaces of a wide range of bacteria and have been hypothesized to be involved in protection, molecular sieving, ion trapping, cell adhesion, surface recognition, and morphogenesis (35). However, only in a few instances have any functions of eubacterial S-layers been rigorously demonstrated, e.g., the protective role of S-layers against Bdellovibrio predation (34) or against nonspecic host defenses (5, 36) and the cell adhesion properties of the S-layer of the sh pathogenic bacterium Aeromonas salmonicida (16, 17, 43). The main cell surface antigenic determinants of A. salmonicida are a smooth layer of lipopolysaccharide (LPS), possessing O polysaccharides of homogeneous lengths, and an S-layer, composed of a single protein subunit (the A-protein) (reviewed in reference 33). Only a small proportion of O-polysaccharide chains penetrate the S-layer, and the majority of LPS molecules remain hidden beneath the S-layer (6, 13). A variety of functions have been experimentally demonstrated for the S-layer of A. salmonicida (reviewed in references 30 and 33); key to the elucidation of function has been the use of isogenic S-layer-negative (S ) mutants. Cultures of A. salmonicida grown at 30 C typically yield mutant cells that lack an S-layer and are avirulent in salmonid sh (29). These S mutants are either unable to synthesize A-protein because of deletions in the N-terminal portion of the corresponding vapA S-layer protein gene (3) or contain natural insertions in the vapA promoter region (23). S-layer function ideally should be recovered by genetic complementation or vapA replacement. However, vapA expression from cloned genes has so far been unsuccessful (8). An alternative to genetic reconstitution is the physical reconstitution of an S-layer on vapA mutants and the subsequent evaluation of recovered functions. Puried S-layer protein subunits from numerous eubacteria are capable of attaching to S cell walls, a process often dependent on the presence of divalent cations, particularly Ca2 (reviewed in references 35 and 42). In the case of the human and animal pathogen Campylobacter fetus, the requirements for reattachment of the S-layer protein to the cell surface of S mutants have been demonstrated (50), but the functional competence of reconstituted cells has yet to be shown. In the presence of divalent cations, puried A-protein can self-assemble into sheets of arrayed layers (18). Also, rough mutants of A. salmonicida lacking LPS O polysaccharide release either A-protein tetrameric units (22) or large sheets of arrayed layers (2, 10) into the culture medium. Reattachment of these released S-layer materials to S mutants has been demonstrated (16, 22). Here, we report the specicity and requirements of the physical reconstitution process, which we
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* Corresponding author. Mailing address: Department of Biochemistry and Microbiology and the Canadian Bacterial Disease Network, University of Victoria, Petch Building, P.O. Box 3055, Victoria, British Columbia V8W 3P6, Canada. Phone: (604) 721-7078. Fax: (604) 7218855. Present address: Department of Veterinary Science, South Dakota State University, Brookings, SD 57007-1396. Present address: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada.

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have approached using different methods, as well as the functional characterization of the S-layer-reconstituted cells. MATERIALS AND METHODS
Bacterial strains. A. salmonicida A449, A450, and A451 are wild-type, virulent strains possessing an S-layer (S ) and full-length LPS O polysaccharide (O ). A450-3 and A449-3 are S O mutants selected by growth at 30 C. A450-1, A451-25, and A451-70 are O mutants which cannot properly anchor their S-layers to the cell surface and therefore release them into the culture medium (Sscr). The following sh pathogens were also used: Aeromonas hydrophila TF7 (S O ), A. hydrophila TF7/U-1 (S O ), Vibrio anguillarum 775, Vibrio ordalii DOM F-3, and Yersinia ruckerii. Escherichia coli B11303, which lacks highmolecular-weight LPS, and Salmonella typhimurium SU453 were used as well. Media and growth conditions. Luria-Bertani broth (LB) or modied LB (MLB) in which NaCl was replaced by modied Davis minimal medium (MDM) [30 mM K2HPO4, 30 mM Na2HPO4, 16.5 mM KH2PO4, 16.5 mM NaH2PO4, 7.5 mM (NH4)2SO4, 0.4 mM MgSO4; pH 7.0] was used throughout. MLB was routinely supplemented with 25 mM D-glucose. Plates of Trypticase soy agar (TSA) or TSA containing 30 mg of Congo red (CR) per ml (TSA-CR) were also used throughout. S strains of A. salmonicida give rise to red colonies on TSA-CR, whereas S strains produce white colonies (28). For some applications, Trypticase soy broth (TSB) and brain heart infusion were also used. The dilution medium used to determine the number of CFU per milliliter by the standard dilution plate method was nutrient broth containing 0.3% Tween 20 (to reduce cell aggregation). Growth of A. salmonicida strains in agitated liquid medium was carried out at 20 C overnight and on plates at 20 C for 3 days. Other strains were grown overnight at 37 C in LB or TSA. Cells grown in liquid media were harvested by centrifugation at 3,000 g at 0 C for 10 min. Routinely, cells were washed in phosphate-buffered saline, pH 7.4 (PBS), and cell suspensions with an optical density at 660 nm (OD660) of 1 were prepared. Cells on plates were gently scraped from the agar surface, then suspended in liquid media or PBS, and subsequently processed as cells grown in liquid media. A-protein extraction and purication. A-protein used for most reconstitution and binding assays was extracted and puried to homogeneity from A. salmonicida A450 by the previously reported method (39). For some applications, low-pH extraction of A-protein using a glycine buffer (pH 2.2) was performed on sodium lauryl sarcosinate-extracted A450 outer membranes (OM), as reported previously (18). Also, A-protein from strain A451-70 was extracted and puried as follows. Cells were grown at 25 C in MLB supplemented with 10 mM sodium citrate, harvested in fresh MDM, and resuspended in MDM supplemented with 1% tryptone and 25 mM D-glucose. Incubation was continued for a further 5 h at 25 C for maximal production of A-protein, and then cells were removed by centrifugation and the culture supernatant was collected. A-protein was precipitated from the supernatant with 50% (wt/vol) ammonium sulfate (at 0 C and in the presence of 0.1 mM phenylmethylsulfonyl uoride [PMSF] and 1 mM EDTA), resuspended in TEPA buffer (20 mM Tris, 1 mM EDTA, 0.1 mM PMSF, and 0.05% NaN3), and dialyzed against TEPA buffer at 4 C. Dialyzed A-protein was dissolved in 1.5 M guanidine HCl and chromatographed at 4 C in a Sephadex G-75 column equilibrated with HEPA buffer (50 mM HEPES [N-2hydroxyethylpiperazine-N -2-ethanesulfonic acid], 2 mM EDTA, 0.1 mM PMSF, and 0.05% NaN3) containing 1.0 M guanidine-HCl. A-protein fractions were pooled, concentrated by ultraltration, and dialyzed against HEPA buffer at 4 C. Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with 12% polyacrylamide slab gels according to the Laemmli method as modied by Ames (1). Alternatively, Nevilles procedure as adapted by Hancock and Carey (24) was used. LPS samples were deproteinated by being incubated with proteinase K at 60 C for 1 h. Protein gels were stained with Coomassie brilliant blue as outlined by Fairbanks et al. (14). LPS gels were stained with silver as described by Tsai and Frasch (45). LPS and LOS purication. Strain A450 (S O ) was grown overnight at 20 C in a 10-liter fermentor holding 5 liters of SP broth (39). Cells ( 100 g [wet weight]) were harvested by centrifugation at 10,000 g, washed twice with 500 ml of deionized water, and then washed sequentially with 500 ml each of 95% ethanol, acetone, and diethyl ether. The resulting pellet was air dried for 30 min. Smooth, or O LPS was extracted by the method of Westphal and Jahn (49), and rough (O ) LPS, or lipooligosaccharide (LOS), was extracted by the method of Galanos et al. (15). Each semipuried fraction was nally puried by gel permeation chromatography through a 200-ml G-75 Sepharose (Pharmacia) column and developed with 0.1 M ammonium acetate buffer at pH 6.4. Elution was monitored by measuring the A302. The puried LPS and LOS fractions were pooled, dialyzed against deionized water, lyophilized, and stored at 20 C. Purity was checked by SDS-PAGE followed by silver staining. Rapid LPS preparation. Small-scale, crude LPS samples containing both LPS and LOS were isolated from different bacterial strains as follows. Bacteria were grown overnight in shaken LB containing 0.5% D-glucose at 20 C, harvested at 5,000 g, and washed once at 0 C with PBS. Lipids were removed by sequential extractions with 10-ml volumes each of acetone and diethyl ether. After 30 min of air drying, the residues were resuspended to 100 mg (dry weight) per ml in PBS, and 150 ml of the suspension was incubated with 20 ml of proteinase K

(Pierce) (10 mg/ml) at 60 C for 2 h. This preparation was estimated to contain approximately 1 mg of LPS per ml and shown to contain only LPS and LOS by SDS-PAGE and silver staining. A-proteinLPS binding. LPS-LOS or LOS preparations (10 g) were dried overnight at 60 C onto wells of Costar microtiter plates. The wells were then blocked by incubation with 200 l of 3% bovine serum albumin (BSA) in PBS at room temperature for 2 h and rinsed with PBS. Various concentrations of A-protein radiolabeled with 125I by using Iodogen (Pierce) (38) in 10 mM Tris HCl (pH 7.0) containing 5 mM CaCl2 or MgCl2 were added to the wells, which were then incubated for at least 2 h to ensure maximum binding. Then the wells were rinsed twice with PBS, and the plates were tapped on absorbent paper to remove excess PBS. The microtiter wells were subsequently severed and assayed for radioactivity by standard gamma radiation counting. Crystallization of A-protein on ultraltration membranes. Highly puried, LPS-free A-protein, derived from culture supernatants of A451-70 (Sscr), was slowly concentrated on hydrophilic ultraltration membranes (Diao YM30; Amicon Co.) at 4 C until crystallization or amorphous precipitation occurred. Details of the method have been described previously (18). S-layer reconstitution with puried A-protein. Bacteria (10 mg [wet weight]) were washed twice in reconstitution buffer (RB) (50 mM Tris HCl, pH 7.4) before being exposed to 0.5 mg of A-protein in 1 ml of RB. Reconstitutions were carried out in 1.5-ml Eppendorf tubes at 22 C for 30 min with gentle agitation. Cells were then pelleted in an Eppendorf microcentrifuge and washed twice with RB. The amount of protein remaining in the supernatant was determined by Lowry assay, and the washed cell pellet was analyzed by SDS-PAGE. For reconstitution at pH 7, 8, or 9, the pH of the RB was adjusted accordingly, and for reconstitution at pH 4, 5, or 6, RB was substituted by 50 mM sodium acetate at the corresponding pH. S-layer reconstitution by coculturing. A450-3 (S O ) was cocultivated with A450-1 (Sscr O ) in TSB or brain heart infusion or on TSA. Suspensions of the two strains to an OD650 of 1 were prepared in PBS and mixed in different ratios. One hundred microliters of each mixture was either used as an inoculum for 5 ml of liquid media or spread on a TSA plate. Tubes were incubated for 24 to 36 h, and plates were incubated for 72 h, at 20 C. The reconstitution cell mixture was processed for viable-cell counts on TSA-CR (to determine the nal A450-3/ A450-1 ratio) as well as for SDS-PAGE. Electron microscopy. Specimens mounted on Formvar-coated grids were negatively stained with an unbuffered saturated solution of ammonium molybdate or with 2% phosphotungstate (pH 7) as previously described (18) and observed in a Philips EM-300 transmission electron microscope at an accelerating voltage of 80 kV. Cell surface hydrophobicity. The following methods were used to determine the relative hydrophobicities of A. salmonicida strains. (i) Salt aggregation test. A 20- l sample of a 1-OD bacterial cell suspension was mixed, on a piece of polystyrene, with 20 l of an (NH4)2SO4 solution at different concentrations, produced by serially diluting a saturated (76-g/liter) solution of (NH4)2SO4 by a factor of 2. The highest dilution at which cell aggregation still occurred was reported as the salt aggregation test titer. (ii) Microbial adherence to hydrocarbons. A 3-ml sample of a 1-OD bacterial cell suspension in PBS was thoroughly mixed with 0.5 ml of hexadecane at 24 C. The mixture was left standing for 20 min, and the OD650 of the aqueous phase was measured to determine the percentage of hexadecane-adhering cells (microbial adherence to hydrocarbons index). (iii) Autoagglutination. Bacterial cell suspensions with an OD of 1 in 5 ml of PBS were left undisturbed in glass tubes at 24 C. At different times, 100- l samples were carefully taken from the surface and diluted 1:10 in PBS, and the OD650 was measured. Assay for phage adsorption. Phage techniques and adsorption assays with the S-layer-specic bacteriophage TP446 were carried out as reported previously (27). Immunoglobulin and porphyrin binding. Immunoglobulin binding assays with whole A. salmonicida cells were performed with 125I-rabbit immunoglobulin G (IgG), as previously described (38). Hemin, protoporphyrin IX (pIX), and CR binding to whole A. salmonicida cells was spectrophotometrically measured as described previously (32). Serum resistance. Bacterial cell suspensions with an OD of 1 in PBS were diluted 1:5 in fresh trout serum. Samples were taken at different intervals to determine the number of CFU per milliliter and the percent survival. Resistance to reduced oxygen species. Suspensions of 3 107 bacteria per ml were prepared in nutrient broth (and exposed to 2 mM H2O2) or in superoxide buffer (and exposed to superoxide generated in situ by the xanthine-xanthine oxidase system [25]). Superoxide buffer was prepared by dissolving 3 mg of xanthine (ICN) in 10 ml of 70 mM phosphate buffer (pH 6.5) at 100 C. After the buffer was cooled to room temperature, EDTA (0.1 mM) was added. Superoxide production was initiated at an approximate rate of 3 mol/min by adding 3 l of xanthine oxidase (ICN) (20 U/ml). Association assays with sh cells. EPC, a cell line from common carp, Cyprinus carpio, and CHSE-214, a line from chinook salmon (Oncorhynchus tschawytscha), were obtained from Microtek Intl. Ltd., Victoria, British Columbia, Canada. Cells were maintained at 15 C in 75-cm2 tissue culture asks (Falcon) holding 50 ml of minimal essential medium (MEM) (Gibco BRL) with 10% fetal bovine serum. Cells from one ask were detached by trypsinization, washed by

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FIG. 1. Reattachment of puried A450 A-protein to A450-3 cells as a function of A-protein concentration and presence of Ca2 . Lanes , 5 mM CaCl2 added to the reconstitution mixture; lanes , reconstitution carried out in the absence of Ca2 . The position of the A-protein band (arrowheads) is indicated.

centrifugation (300 g for 10 min at 22 C) in fresh MEM, suspended in MEM-plate medium (MEM with 2% fetal bovine serum, 10 mM HEPES, 1,000 U of penicillin per ml, 1 mg of streptomycin per ml, and 2.5 mg of amphotericin B per ml), and dispensed into a 24-well plate at 106 cells per well. After an overnight incubation at 15 C, the culture medium was replaced with 1 ml of PBS, and 100- l aliquots of the bacterial cell suspension at an OD650 of 1 were added so that all the wells in the plate could be processed simultaneously at the end of the experiment by shaking off the well supernatants and washing the plate three times with PBS in a wash bottle. Cells were air dried, xed in methanol, and stained with Giemsa stain. Well bottoms were cut with a cork-boring machine and mounted on glass slides for light microscopy. The average number of bacteria per sh cell was determined by direct counts for at least 100 cells per well bottom. Survival within trout M . Survival of A. salmonicida within isolated head kidney macrophages (M ) from rainbow trout (Onchorynchus mykiss) was determined in medium containing 5 mg of gentamicin per ml as described previously (20). Since A. salmonicida is cytotoxic for M , the results are presented as percent survival per living M , an index which incorporates only the number of nonlysed M in the assay tubes (20).

RESULTS Self-assembly and reattachment of A-protein to whole cells. When in solution, puried A-protein had the tendency to spontaneously form multimers. Previous measurements, using sedimentation analysis and high-performance liquid chromatography molecular sieving, indicated that up to 30 to 40% of the puried A-protein apparently exists as aggregates with a predominant Mr of 100,000 to 180,000 (31, 39). Although some reattachment of puried A450 A-protein to the A450-3 A. salmonicida strain (S O ) occurred in the absence of Ca2 , it was clearly enhanced by the addition of Ca2 over a wide range of A-protein concentrations (0.02 to 1.0 mg/ml) (Fig. 1). Electron microscopy showed that S-layer sloughs displaying typical tetragonal arrangements were present only when A450-3 cells were reconstituted in the presence of Ca2 and with A-protein concentrations of 0.5 mg/ml (Fig. 2a). Regular arrays were obscured by the presence of A-protein aggregates and/or multilayered sloughs (distinguished by their darker staining and loss of ne structural detail) (Fig. 2b), which were formed in direct proportion to the A-protein concentration in the range of 50 to 500 g/ml. At a concentration of 0.5 mg/ml, regular arrays were completely obscured by the massive aggregation of A-protein at the cell surface. Thus, Ca2 affected both formation of normal tetragonal arrays from A-protein in solution and enhanced reattachment of puried A-protein to the cell surface of A450-3 cells. With respect to the specicity of the reattachment process, A-protein reattached only to A. salmonicida cells which already possessed either an S-layer or an exposed O-polysaccharide layer. Puried A-protein was unable to reattach to A. salmonicida O mutants or cells of the heterologous strains tested, even if they possessed O polysaccharide (Table 1). The efcient reattachment of A-protein to the S O A450 strain implied that multiple S-layers may have formed on the existent

FIG. 2. Electron micrographs of negatively stained A450-3 cells reconstituted with A-protein in the presence of 5 mM Ca2 . (a and b) S-layer sloughs from cells reconstituted with 60 g or 0.2 mg of A-protein per ml, respectively. Areas with superimposed S-layers (S) or A-protein aggregates (A) are indicated (arrows). Bars, 0.1 m.

(naturally assembled) S-layer. While the actual formation of multiple layers at the surface of reconstituted A450 or A450-3 cells was not further explored through electron microscopy, functional characterization (see below) indicated that excess A-protein was present in a rather disorganized state. Self-assembly and reattachment of A-protein in the absence of Ca2 . Reattachment of A-protein to A450-3 was less efcient in the absence of Ca2 than in its presence (Fig. 1). Moreover, in negatively stained preparations of A450-3 cells reconstituted in the absence of Ca2 , we observed a large number of ring-shaped units (Fig. 3a) but no regular arrays. These units were very similar to the ring-shaped tetrameric units previously found in preparations of A450 cells either grown in a Ca2 -decient medium or treated with 0.5 M ethylene glycol-bis( -aminoethylether)-N,N,N ,N -tetraacetic acid (EGTA) (18) and were thus considered to be stable tetrameric oligomers of A-protein that had bound to A450-3 cells. Because the ring-shaped units were formed in the virtual absence of Ca2 , we concluded that Ca2 was not required at this level of assembly. Moreover, A-protein puried from A451-70 (Sscr), when concentrated on ultraltration membranes in divalent-cation-decient HEPA buffer, was still able to form macroscopic semicrystalline sheets (Fig. 3b). In suspension they resembled cellophane sheets, and upon drying they were rigid and refractile and fractured along straight lines. Negative

VOL. 177, 1995 TABLE 1. Reattachment of A450 puried A-protein to different cell templates
Characteristic of cell surface Strain or species O polysaccharide S-layer Reattachment of A-proteina

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A. salmonicida A449-3 A450 A450 extractedb A450-3 A451 extractedb A451-25c A451-70c A. hydrophila TF7 TF7/U-1 E. coli S. typhimurium V. anguillarum

Secreted Secreted Truncated

a Assayed by SDS-PAGE. , A-protein reattached; , very small amount of A-protein reattached; , no A-protein reattached. b A-protein in these cells was extracted by the low-pH extraction method (38). c Rifampin at a nal concentration of 200 g/ml was added to block the synthesis of secreted A-protein.

staining of sonicated specimens indicated that these sheets were composed primarily of tetragonal arrays (Fig. 3c) which displayed a previously described pattern formed by independent (not interlocked) A-protein morphological units (18). Thus, in the absence of Ca2 , A-protein subunits self-assembled into ring-shaped units, which in turn were able to reattach to O cell templates, group into loose arrays, and in some instances form macroscopic semicrystalline sheets. Reattachment of puried A-protein to A. salmonicida A450-3 (S O ) under dened conditions. The adsorption of puried A-protein to A450-3 cells under various conditions of ion type and concentration, pH, time, and temperature was extensively investigated (Table 2). Again, Ca2 enhanced binding of Aprotein to the surface of A450-3 cells at different temperatures and pHs, as well as in the presence of Tween 20. Mg2 also had an enhancing effect, albeit weaker than that of Ca2 (Table 2). Monovalent ions had no apparent positive or negative effect, suggesting that the enhancing effect of Ca2 was not due simply to nonspecic charge shielding. Nonetheless, Ca2 was clearly not essential for reattachment. The involvement of ionic interactions between A-protein and the smooth LPS surface layer of A450-3 was strongly supported by the reconstitution results obtained at different pHs. At a low pH, A-protein adsorption was efcient and largely Ca2 independent. Adsorption at pH 5, which corresponds to the pI of A-protein, could not be assayed, as the protein precipitated. At a pH of 6, the enhancing effect of Ca2 was clearly evident, and at pH 9, Ca2 was essential for reattachment (Table 2); these results all indicate that electrostatic repulsion and charge neutralization were important to the A-protein reattachment process. Tween 20 did not inhibit A-protein adsorption in the presence or absence of Ca2 (Table 2), indicating that hydrophobic interactions play a negligible role. Had hydrophobic interactions been largely responsible for A-protein adsorption, the protein should have reattached more efciently to the hydrophobic surfaces of the O strains A451-25 and A451-70 (Table 1). The interactions between adsorbed A-protein and the OM appeared to be readily dissociable, since upon extraction with sodium lauryl sarcosinate, OM prepared from reconstituted

FIG. 3. Micrographs of A-protein self-assembly products formed in the absence of Ca2 . (a) A-protein aggregate in the proximity of cells reconstituted with 60 g of A-protein per ml. Bar, 0.1 m. (Inset) Enlargement of one of the aggregates showing distinct ring-shaped units. Bar, 25 nm. (b) Piece of a semicrystalline sheet of A-protein formed on an ultraltration membrane, as seen through a phase-contrast light microscope. Bar, 0.1 mm. (c) Regular arrays composing the semicrystalline sheets shown in panel b. Note the discontinuity of the arrays and the apparent holes (arrowheads) within them. Bar, 50 nm.

A450-3 cells lost a large portion of the reattached A-protein. However, the remaining OM-bound fraction (10 to 20%) of the reattached protein was resistant to a further low-pH extraction and therefore was assumed to be tightly attached (not shown). Reattachment was rapid, being essentially complete in less

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TABLE 2. Reattachment of A450 A-protein to A450-3 cells under various conditionsa


Rb Condition In absence of Ca2 In presence of Ca2

Reagent added None (control) 1 mM Ca2 5 mM Ca2 10 mM Ca2 10 mM Li 10 mM Mg2 0.05% Tween 0.2% Tween 10 mM EDTA 10 mM EGTA 0.1 M NaCl 0.5 M NaCl Temp ( C) 4 22 30 37 Time (min) 5 20 30 60 pH 4 5 6 7 7.4 8 9

FIG. 4. Kinetics of 125I-A-protein binding to high-molecular-weight LPS puried from A. salmonicida A450. Radiolabeled A-protein (specic activity, 5.5 104 Ci/ mol) was added, at various concentrations, to 10 mol of LPS coating each well of a 96-well microtiter plate, in 10 mM Tris HCl (pH 7.0) and 5 mM CaCl2 at room temperature. After incubation, wells were assayed for radioactivity. (Inset) Double-reciprocal plot from which the Kd of A-proteinLPS binding was determined.

PPTc

PPT

a Except for the indicated changes in the conditions of the assay, the standard reconstitution was performed in the presence of 5 mM Ca2 at 22 C and pH 7.4 for 30 min. b R, reattachment of A-protein determined by SDS-PAGE. , no reattachment; , little protein reattached (less than the wild-type level); , reattachment to levels similar to that of the wild type; , reattachment to levels 1.5 to 2.0 times higher than that of the wild type; , reattachment to levels 2 times higher than that of the wild type. c PPT, A-protein precipitated in the pH 5 buffer in the absence of added A450-3 cells.

than 5 min, and to some extent temperature dependent, since a temperature increase in the interval of 4 to 30 C roughly correlated with an increase in A-protein adsorption (Table 2). Binding of puried A-protein to puried LPS. To further examine the A-proteinLPS interaction, an in vitro reconstitution assay using 125I-labeled A-protein and highly puried LPS or LOS immobilized in microtiter wells was developed. A number of parameters were established for this highly sensitive assay: buffers (PBS, 10 mM Tris HCl, or 10 mM HEPES at pH 7.0), receptor concentration (10 mg of LPS per well), incubation time ( 2 h), effects of divalent cations (Mn2 Ca2 Mg2 Fe2 ), and pH (pH 6 to 9). In addition, the assay was partially inhibited ( 50%) by 0.1 M EDTA or EGTA or by the nonionic detergent Tween 20. However, the assay was unaffected by up to 5% BSA; in fact, 3% BSA was used as a blocking agent in the assay. The shallow sigmoidal shape of the saturation curve (Fig. 4) indicated that A-protein binding was slightly cooperative. The double-reciprocal plot of the higher A-protein levels of this curve (Fig. 4, inset) indicated that the apparent afnity of

A-protein for high-molecular-weight LPS of strain A450 was fairly high (Kd, 1.55 10 7 M). LPS specically acted as the receptor for A-protein, since puried A. salmonicida LOS preparations were entirely ineffective (not shown), indicating that the O-antigen portion of LPS was the receptor for A-protein. To examine the specicity of the receptor, a rapid method of purifying LPS-LOS from a variety of bacteria which obviated the necessity of laborious LPS purication protocols was developed (see Materials and Methods, Rapid LPS preparation). Binding of radiolabeled A-protein to the various LPSLOS preparations showed a high level of specicity for the LPS of A. salmonicida (Fig. 5). LPS-LOS preparations from other bacteria, including several pathogenic Vibrio species and the S-layer-harboring strain A. hydrophila Ah65, were entirely ineffective as A-protein receptors, as was the LOS of the O A. salmonicida strain A450-1. Thus, there exists a highly specic, high-afnity binding interaction between A-protein and the O polysaccharide of A. salmonicida, which correlated well with the specicity of A-protein attachment observed in the previous whole-cell assays (Table 1). Also in agreement with the results obtained with whole cells (Table 2), this binding was pH dependent. A-protein binding to highly puried A. salmonicida LPS was affected by dened chemical modications introduced in the A-protein molecule (Table 3). Lastly, proteolytic digestion of A-protein with either trypsin, chymotrypsin, or proteinase K completely eliminated binding, whether assayed directly or competitively by using proteolysis products. Physical reconstitution by coculturing. To more effectively reconstitute a highly ordered S-layer on S O cells, a coculture method which took advantage of the natural S-layer assembly process in vivo was established. A. salmonicida A450-1 (Sscr) releases large, highly ordered S-layer sheets (Fig. 6a) which were readily adsorbed onto the surface of A450-3 cells (S O ) (Fig. 6b and c). When the two strains were grown together in liquid or solid media, A450-1 acted as an S-layer donor and A450-3 acted as a receiver. We observed that a

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A. SALMONICIDA S-LAYER RECONSTITUTION TABLE 3. A-protein binding to puried LPS and effects of A-protein modications on binding
Reagent(s)a

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% Bindingb

A-protein alone ......................................................................... 100 A-protein plus: 5 mM Ca2 ............................................................................ 200 5 mM Mg2 ............................................................................ 180 5 mM Mn2 ........................................................................... 220 5 mM Fe2 ............................................................................. 150 Various sugarsc ...................................................................... 76116 Iodoacetate............................................................................. 2.8 Acetic anhydride.................................................................... 8.8 Chymotrypsin ......................................................................... 1.0 Trypsin .................................................................................... 1.0 CNBr....................................................................................... 4.0 Formaldehyde ........................................................................ 2.0 Chloramine T......................................................................... 40
a A-protein was allowed to react with the various modication reagents before binding to LPS was assayed. A-protein chemical modications were carried out as described previously (38). b Percent binding was determined by quantication of A-protein in the assays microtiter plates (refer to Materials and Methods). c The following sugars were tested at a concentration of 0.1 M: -D-glucose, -D-glucose, L-rhamnose, D-mannose, D-manosamine, NAc-D-manosamine, Dgalactose, and NAc-D-glucosamine.

FIG. 5. Specicity of binding of A-protein to immobilized LPS-LOS. A-protein binding to LPS-LOS puried from O A. salmonicida strain A450 (S ) (E) or A450-3 (S ) (F) as well as to LOS puried from strain A450-1 (Sscr O ) () is shown. The binding to the LPS-LOS puried from other gram-negative bacteria (V. anguillarum, V. ordalii, Y. ruckerii, S. typhimurium, and E. coli) was essentially the same as that shown for A. hydrophila Ah65 (). (Inset) Silverstained SDS-PAGE gel of the LPS-LOS of the A. salmonicida strains tested. Lanes: 1, A450; 2, A450-3; 3, A450-1.

change in the original A450-3-to-A450-1 cell ratio in the inoculum had an effect upon both the nal amount of A-protein associated with the cell reconstitution mixture and the nal ratio of the two cell types (Fig. 6c). The best reconstitution was obtained when carried out on TSA plates inoculated with an initial A450-3/A450-1 ratio of 1 or 1.5. Also, it was possible to reconstitute A450-3 cells by suspending them in supernatants of A450-1 cultures. However, this method of reconstitution in a liquid phase was not always effective, as judged by the small amount of reattached A-protein detected by SDS-PAGE in some reconstitution experiments (not shown). Figure 6c also shows that coculturing in liquid media was not as effective as coculturing on plates, suggesting that the liquid system imposed some constraints on the reconstitution process. We have hypothesized previously (16) that a major constraint may be the tendency of released S-layers in suspension to readily form double layers that lack polarity (10). Functional characterization of A-protein-reconstituted cells. Cells of A450-3 (S O ) reconstituted by reattachment of puried A-protein bound pIX, hemin, and the S-layer-specic phage TP446, in proportion to the amount of protein previously adsorbed (Table 4). Binding was improved when reconstitution was performed in the presence of Ca2 . However, when IgG binding (which is an array-dependent function) was assayed, only a small fraction (5.6%) of the wild-type binding level was present in reconstituted A450-3 cells, and this was not improved by the addition of Ca2 (Table 4). Curiously, the macroscopic semicrystalline sheets of A451-70 A-protein formed on ultraltration membranes in the absence of Ca2 possessed a signicant IgG binding activity (not shown), indicating that these sheets displayed a better-organized interface than A-protein-reconstituted cells. On the other hand, reconstituted A450 partially lost all four binding abilities in rough proportion to the amount of reattached Aprotein (Table 4).

Functional characterization of cells reconstituted by coculturing. Since coculturing reconstitution on plates was simple, consistently achieved, and not subject to a haphazard adsorption of A-protein, it was used for further functional characterization. Several known, S-layer-mediated functions relevant to pathogenesis were tested either on the reconstitution cell mixture or, more specically, on reconstituted A450-3 cells, differentiated on TSA-CR plates. S A. salmonicida is far more hydrophobic than S (12, 46), and this has been implicated in adherence to host cells (37, 43). The reconstitution cell mixture with an average A450-3/A450-1 ratio of 1.3 0.24, and possessing a relative hydrophobicity higher than the average hydrophobicity of the two strains involved (Table 5), was as autoaggregative as A450 or A450-1 alone (Fig. 7) and highly adherent to EPC and CHSE-214 sh cell lines as well (Table 5). Similarly reconstituted cells were previously shown to bind the heme analog CR and to adhere to M (16) (data included in Table 5). It was interesting to discover that these reconstituted cells also exhibited enhanced survival within trout M (Table 5). Thus, they had regained the ability not only to invade M but also to resist M killing mechanisms. However, the reconstituted S-layer was unable to provide A450-3 cells with extended protection against complement-mediated killing (Table 5). The protective role of the A. salmonicida S-layer against reduced oxygen species has recently been studied (21). Interestingly, the reconstituted S-layer had protective effects against reduced oxygen species similar to those of the wild-type layer (Table 5). Also, we have recently proposed that the S-layer of A. salmonicida is involved in the uptake of streptonigrin (SNG) and chloramphenicol (CM), which is manifested in an extremely high level of sensitivity of S strains to these intracellularly acting antibiotics (19). Data from these studies show that reconstituted A450-3 did not recover a high level of sensitivity to SNG or CM (included in Table 5). DISCUSSION The discovery that spontaneous (26) or transposon-induced (2) mutations leading to a change from LPS to LOS give rise to

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FIG. 6. Physical reconstitution of A450-3 by coculturing with A450-1. (a) Electron micrograph of a typical S-layer sheet released by A450-1. Bar, 0.1 m. (b) Cell border of a cell in the reconstitution mixture showing a hemispherical S-layer slough. Since S-layer sheets are never hemispherical in A450-1, this was assumed to be an A450-3 cell that had picked up S-layer material produced by A450-1. Bar, 0.1 m. (c) Coomassie brilliant blue-stained SDS-PAGE gels of whole-cell lysates from different reconstitution cell mixtures obtained after cogrowth of the two strains in brain heart infusion or TSB or on TSA. The ratios of A450-3 to A450-1 cells after cogrowth are shown above the gels. Cell mixtures were inoculated with the following A450-3/A450-1 ratios: lanes 1, 0.5; lanes 2, 1.0; lanes 3, 1.5; and lanes 4, 2.0. The position of the A-protein band (arrowheads) is indicated.

phenotypically S A. salmonicida strains that release A-protein as tetragonally arrayed double S-layers (10) led to the assumption that LPS was involved in tethering the A. salmonicida S-layer to the bacterial OM. The results presented here clearly conrmed this assumption; we found that the primary S-layer constituent, A-protein, binds to the A. salmonicida LPS O antigen specically and with high afnity. In spite of differences

in the A-protein concentrations used, the reattachment of Aprotein to viable, S-layer-decient cells was remarkably similar to the in vitro binding of A-protein to puried LPS. These similarities extended to the LPS receptor, the specicity of the interaction, and the effect of divalent cations. Binding of Aprotein to LPS appeared to involve ionic interactions between essential amino groups in the A-protein and acidic residues in

TABLE 4. Functional characterization of A-protein-reconstituted cells with respect to pIX, hemin, and immunoglobulin bindinga and S-layer-specic bacteriophage TP446 adsorption
Strain A-protein added for reconstitution Bound pIX ( g) Bound hemin ( g) mol of bound rabbit IgG (10 11) Phage adsorbed (%)

A450

None 0.2 mg/ml 0.5 mg/ml 0.2 mg/ml 0.5 mg/ml None 0.2 mg/ml 0.5 mg/ml 0.2 mg/ml 0.5 mg/ml

5 mM Ca2 5 mM Ca2

2.18 1.61 1.26 1.40 1.35 1.19 1.65 2.25 1.99 2.15

0.12 0.08 0.15 0.10 0.28 0.36 0.02 0.17 0.07 0.03

1.03 0.89 0.88 0.68 0.77 0.21 0.32 0.52 0.39 0.68

0.27 0.01 0.32 0.08 0.09 0.20 0.19 0.15 0.01 0.02
10
10

7.18 4.32 3.88 3.18 2.86 0.11 0.32 0.40 0.36 0.40

0.41 0.00 0.11 0.08 0.08 0.02 0.00 0.00 0.00 0.00

99.9 99.7 97.7 65.0 59.0 7.8 38.0 51.5 59.1 44.6

0.3 0.5 0.7 2.9 13.0 2.7 17.1 6.6 6.5 3.4

A450-3

5 mM Ca2 5 mM Ca2

a Binding assays were performed with the following amounts of available ligands: 3 g of pIX or hemin or 4 experiments and are presented as means standard deviations from the means.

mol of rabbit IgG. Data are results from duplicate

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A. SALMONICIDA S-LAYER RECONSTITUTION TABLE 5. Functional characterization of cocultured reconstituted cells, either as a reconstitution cell mixture or as reconstituted A450-3 cellsa
No. of adhering bacteria perb: Susceptibility (% survival)d to: CR bound ( g)c Serum (3 h) SNG (3.5 h) CM (6.5 h) H2O2 (5 h) (10
4

2691

Strain

MATH index

SAT titer

100 M

EPC cell

CHSE-214 cell

SPO (3 h)

Killing by m (10 h)

A450 A450-3 A450-1 Rec. mix. Rec. A450-3


a

49.5 28.3 29.0 46.2

16 2 16 8

525 37 102 597

51 3.5 23 58

47 2.4 3.3 29

1.0 0.2 0.4 0.7

25.0 4.1 0.003 2.0

0.004 5.3 0.33 2.9

1.1 88.5 25.5 89.0

1.2 0.2 1.0 1.0

5.3 0.03 1.0 7.6

8.9 0.6 0.2 6.0

Data presented are the averages of duplicate results or results from at least two independent experiments. Standard deviations have been omitted for clarity. Abbreviations: MATH, microbial adherence to hydrocarbons; SAT, salt aggregation test; SPO, superoxide; Rec. mix., reconstitution cell mixture with an average A450-3/A450-1 ratio of 1.3 0.24; Rec. A450-3, reconstituted A450-3 cells selectively differentiated on TSA-CR plates during viable-cell counts. b Adherence to sh cells measured in 3-h assays. c A 1.3- g sample of CR was added to 1 ml of a bacterial cell suspension with an OD650 of 1. d Susceptibility to the different agents shown was evaluated through viable-cell counts at the times indicated in parentheses and compared with counts at time zero. The following concentrations were used: 80% serum, 1 g of SNG per ml, 100 g of CM per ml, 2 mM H2O2, or 3 mol of SPO per min ml.

the LPS. Primary amine nucleophiles were apparently involved, since both iodoacetate and acetic anhydride inactivated binding activity. However, the O-antigen portion of the A. salmonicida LPS does not contain acidic residues (41), and the acidic LOS fraction did not act as an A-protein receptor. This raises the possibility of a unique A-proteinO-polysaccharide binding site that both recognizes a complex carbohydrate epitope (accounting for the failure of monosaccharides to block binding) and is conformed only upon proper folding of the protein (accounting for the release of native or reattached A-protein by low pH and for the failure of proteolytic fragments to bind, or inhibit binding of A-protein, to LPS). It is possible that at low pH a conformational shift occurs in the A-protein structure, masking or disrupting this putative LPSbinding domain. While most A-protein subunits may interact with O-polysaccharide chains, there exists a small fraction ( 10%) of the reattached A-protein that was able to establish tight interactions (not dissociable by low pH) with the OM. The nature of these stronger interactions has not yet been dened, but they appeared to be different from the dissociable LPSA-protein interactions that held the majority of S-layer subunits on the

FIG. 7. Autoaggregation proles of A450 (F), A450-3 (E), A-450-1 ( ), and the reconstitution mixture (X) with an average A450-3/A450-1 ratio of 1.3.

cell surface. We have hypothesized that these tightly OMassociated subunits interact with OM proteins (most likely porins) or are inserted in the OM (19). Other gram-negative bacteria whose S-layers specically interact with LPS also require Ca2 for S-layer assembly and/or attachment (7, 47, 48, 50). For A. salmonicida, Ca2 played a dual role. First, Ca2 was essential in organizing the reattached protein into interlocked, normal tetragonal arrays. In the absence of both LPS and Ca2 , A-protein was quite capable of spontaneously assembling into oligomers, noninterlocked loose arrays, and even macroscopic semicrystalline sheets. However, only in the presence of Ca2 were normal interlocked arrays assembled, indicating that in agreement with previous results (18), the nal subunit-subunit interaction responsible for the formation of the two morphological units characteristic of normal tetragonal arrays (10) was mediated by Ca2 . Ca2 may be essential in the formation of cationic bridges between the A-protein domains that form the minor morphological unit (18) or in the induction of a conformational state in the A-protein conducive to formation of the interlocked tetragonal array or both. Ca2 has also been shown to effect the in vivo assembly of S-layer protein subunits of Azotobacter vinelandii into a tetragonal array (11). Second, Ca2 played an enhancing but nonessential role in the reattachment of A-protein to LPS O polysaccharide. Because our previous (18) and present ndings have clearly shown that Ca2 is involved in the interaction within S-layer morphological units more than in S-layerLPS interactions, the question was thus raised as to whether Ca2 makes A-protein a better ligand for the LPS receptor (by inducing an assembly-procient conformation) or whether enhanced reattachment was a natural result of the Ca2 -mediated organization of A-protein into normal arrays. Presently, none of these alternate situations can be strictly excluded on the basis of available experimental evidence. The functional characterization of reconstituted cells and the clear relationship between structural S-layer features and functional competence were of special interest. For instance, excess A-protein adsorbed onto the cell surface of A450 (S O ) reduced binding competence (Table 4). This suggested that (i) the reattached protein did not acquire a native arrangement, (ii) excess A-protein most likely reattached in a haphazard fashion, and/or (iii) specic binding sites on the native S-layer were blocked. Apparently, these binding sites must have a dened orientation in the native S-layer (presumably at the outer face of the layer) to be optimally competent. Thus,

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FIG. 8. Schematic representation of the physical S-layer reconstitution of A. salmonicida. Different conditions of reconstitution (e.g., type of reconstitution material used and presence or absence of a reconstitution template or Ca2 ) lead to different levels of organization and functional competence. Monomeric A-protein (center) may interact with itself to form tetrameric intermediate units directly on a template or in solution. Depending on the availability of Ca2 , the conformation of the units (and consequently of the arrays that they form) might be different. Monomeric and tetrameric A-protein may be rapidly bound to the randomly immobilized LPS template, precluding formation of arrays. Apparently, a native LPS layer on the cell surface promotes the formation of S-layer arrays in the presence of Ca2 . The best reconstitution was achieved with preassembled arrays bound to a smooth-LPS cell surface template.

the orientation and order of the layer must be important for optimal binding. The importance of a correct S-layer orientation in the establishment of proper S-layerOM interactions has also been previously emphasized (10, 16). Most likely, A450-3 (S O ) cells reconstituted with puried A-protein were not optimally competent in binding porphyrins or phage because of the haphazard accumulation of the reattached Aprotein. The poor order of the reattached A-protein was further suggested by its inability to bind immunoglobulins. The proposed IgG binding sites, formed by the ordered interaction of several A-protein subunits (38), were not efciently conformed on A-protein-reconstituted A450-3 cells. These ndings are in agreement with previous ones showing that puried A-protein adsorbed onto latex beads was unable to mediate high-level adherence to M , whereas beads coated with puried S-layer sheets were highly adherent (17). Interestingly, free S-layer sheets released by the Sscr strain A449 TM5 were capable of binding collagen type IV, laminin, and bronectin (9, 44), and particularly, binding of collagen type IV appeared also to depend on the presence of a natively assembled S-layer (44). Therefore, it was not surprising that the most structurally and functionally correct reconstitutions were achieved by allowing an in situ transfer of preformed S-layers to S O cells by the coculturing technique described here. Similarly, S-layer reconstitution in Azotobacter vinelandii was achieved only with preformed S-layers (4). A450-3 cells reconstituted by coculturing were competent in many of the functions tested, e.g., porphyrin binding, adherence to host cells, resistance to reduced oxygen species, and even resistance to M killing (Table 5). However, not even these cells, reconstituted with preassembled S-layers, were competent with respect to serum resistance and susceptibility to SNG and CM. Apparently, a native S-layer arrangement was essential to confer protec-

tion from complement-mediated killing. S-layer-reconstituted cells may have had uncovered zones, or major S-layer faults, on which complement in trout serum, which has been demonstrated to be particularly lytic against A. salmonicida (20), could x and form membrane attack complexes. Also, a native assembly appeared necessary for mediating an enhanced cell permeation by SNG and CM, since reconstituted cells were not highly susceptible to these antibiotics (19). While we have emphasized S-layer functions relevant to pathogenesis, it should be noted that there are other functions relevant to the survival of A. salmonicida in the aquatic environment, e.g., S-layer-mediated resistance to predation by Bdellovibrio bacteriovorus (34) or long-term survival in river sediments (40). Although these were not assayed here, we still believe that the functional characterization of reconstituted A. salmonicida cells reported here is the rst of its kind in S-layer research, since most S-layer reconstitution studies have been approached exclusively from a structural perspective (22, 35, 42, 50). A summary of our ndings is shown in Fig. 8. Puried Aprotein monomers may bind directly to LPS and/or self-assemble into oligomers of a dened conformation, depending on the availability of Ca2 . In turn, oligomers may also bind to LPS and/or assemble into loose or interlocked arrays, again depending on the availability of Ca2 . Finally, the degree of order achieved (either at the monomeric, oligomeric, or supramolecular level) may inuence the efciency of reconstitution and the functional competence of the reconstituted products. When assembled either in highly ordered interlocked arrays or in loose arrays of independent units, reconstituted S-layers are functionally more competent than A-protein directly reattached to LPS, or a cell surface template, in a less ordered manner.

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From a functional perspective, the physical reconstitution of S A. salmonicida had signicant limitations directly related to dened structural constraints of the reconstitution process. Nevertheless, in the absence of genetic reconstitution, physical reconstitution has proved to be a very useful tool in studying the functions of the A. salmonicida S-layer and identifying relevant S-layer structure-function relationships.
ACKNOWLEDGMENTS

21.

22. 23. 24.

These studies were supported by grants to W.W.K. from the Natural Sciences and Engineering Research Council of Canada (NSERC) through the Canadian Bacterial Diseases Network. The technical assistance of T. Ainsworth, J. Winchester, K. Witthal, and T. Yu is gratefully acknowledged, as is the bacteriophage expertise of E. E. Ishiguro.
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