Reassembly of a Fimbrial Hemagglutinin from Pseudomonas

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1985, p /85/ $02.00/0 Copyright ( 1985, American Society for Microbiology Vol. 50, No. 3 Reassembly of a Fimbrial Hemagglutinin from Pseudomonas solanacearum after Purification of the Subunit by Preparative Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis DAVID H. YOUNG,t* WILLEM P. C. STEMMER, AND LUIS SEQUEIRA Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin, Received 31 December 1984/Accepted 17 June 1985 Distilled water homogenates of Pseudomonas solanacearum B1, a highly fimbriated strain, strongly agglutinated human group A erythrocytes. The fimbriae and hemagglutinating activity were precipitated from the crude extract with 1% acetic acid, redissolved at ph 10, and precipitated again with 20 mm CaC12 at ph 6.0. Ca2+, Mg2+, and Zn2+ had similar ability to precipitate the fimbrial hemagglutinin, but Na+ and K+ were much less effective. The fimbrial protein in the precipitate was purified to homogeneity by preparative gel electrophoresis in sodium dodecyl sulfate. The major protein band was eluted, and sodium dodecyl sulfate was removed by chromatography on ion retardation resin (AG 11A8) in 6 M urea. After dialysis against 10 mm sodium acetate (ph 4.5) to remove the urea, the protein reassembled to yield long fibers. These fibers were identical to fimbriae in the crude extract in diameter (6 nm) and in their ability to cause hemagglutination. The purified fimbriae contained no carbohydrates and were similar to other bacterial fimbriae in amino acid composition, with hydrophobic amino acids comprising 41.8% of the total. The introduction of certain avirulent strains (Bi, for example) of the bacterial wilt pathogen Pseudomonas solanacearum E. F. Sm. into tobacco leaves results in attachment and envelopment of the bacteria by the host mesophyll cell walls, followed by induction of a hypersensitive response (20). Virulent strains do not attach or induce the hypersensitive response on host cells, which suggests that attachment is important in triggering the host's defense mechanisms. Previous studies have focussed on the interaction of the rough lipopolysaccharide of Bi cells with a bacterial agglutinin that is present on the host cell walls (3, 14). The possible involvement of fimbriae, which mediate bacterial adhesion to many surfaces (7), has not been examined in this system. Fimbriae of 4.9 to 6.0 nm in diameter in P. solanacearium were first described by Fuerst and Hayward (6). Stemmer and Sequeira (20a) have reported that the avirulent Bi strain of this bacterium is highly fimbriated but that its virulent parental strain, K60, produces very few fimbriae. Because these two strains also differ in their ability to attach to plant cell walls (20), it seemed important to purify the Bi fimbriae to examine their properties and possible role in the process of attachment. In this report we describe the use of preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to isolate the fimbrial protein subunit and the reassembly of these subunits to whole fimbriae. We describe a novel means to purify fimbriae to homogeneity with retention of their hemagglutinating activity. MATERIALS AND METHODS Bacteria and growth conditions. An avirulent, afluidal variant (Bi) of P. solanacearum (11) was obtained from A. Kelman. Bacteria were grown as a lawn in petri dishes at 28 C * Corresponding author. t Present address: Research Division, Rohm & Haas Company, Spring House, PA for 60 h on the agar medium described by Kelman (10) except that triphenyl tetrazolium chloride was omitted. SDS-PAGE. SDS-PAGE was performed on 12.5% gel in a Bio-Rad Protean double slab electrophoresis cell (Bio-Rad Laboratories, Richmond, Calif.) at 15 C. The system described by Anderson et al. (1) for separating proteins in the low-molecular-weight range was used. Preparative gels (6 by 160 by 140 cm) were run at 45 W for 14 h (the marker dye, bromophenol blue, reached the end of the gel after ca. 11 h). The upper buffer was replaced with fresh buffer at 3-h intervals. Analytical gels (1.5 by 160 by 140 cm) were run at 20 W until the marker dye reached the end of the gel (ca. 8 h), and the upper buffer was changed after 4 h. Gels were stained for protein with Coomassie brilliant blue R250 (13). The periodic acid-schiff reagent (5) was used to detect carbohydrate. Purification of the fimbrial hemagglutinin. Bacteria were scraped from the surface of 50 petri dishes (8.5 g [wet wt]) and suspended in 50 ml of distilled water. A crude extract was prepared by homogenizing the suspension in an ice bath then removing the cells by centrifugation at 10,000 x g for 15 min at 4 C. Glacial acetic acid (0.5 ml) was added to the supernatant with stirring in the cold. After 15 min, the material that precipitated was pelleted by centrifugation and suspended in 25 ml of cold distilled water. The ph of the suspension was adjusted to 10.0 by dropwise addition of 0.2 N NaOH, and the preparation was stirred in the cold until the precipitate had dissolved. After adjusting the ph to 6.0 with acetic acid, CaCl2 (2 M) was added to a final concentration of 20 mm, and the preparation was stirred in the cold for 15 min. Then the precipitated material was collected by centrifugation. The pellet was dissolved in 2 ml of SDS-PAGE sample buffer (0.139 M Tris, 1% SDS, adjusted to ph 7.8 with glacial acetic acid). Then a sample was labeled with fluorescamine, which fluoresces strongly after reacting with amino groups (21). For this purpose, 0.2 ml of a 5-mg/ml solution of

2 606 YOUNG ET AL. APPL. ENVIRON. MICROBIOL. fluorescamine (Sigma Chemical Co., St. Louis, Mo.) in dimethyl sulfoxide was added to 0.4 ml of sample with vigorous stirring on a Vortex mixer. After incubating at room temperature overnight, the labeled and unlabeled samples were combined, and then 300 mg of sucrose and 160 RI of a 1-mg/ml solution of bromophenol blue in sample buffer were added. After preparative SDS-PAGE of the sample as described above, the gel was viewed under UV light (310 nm) to visualize the separated proteins, and the major protein band (Rf 0.58) was excised as a narrow slice, approximately 4 mm wide. The gel slice was homogenized by squeezing it through a syringe, and the protein was eluted by shaking the gel homogenate for 10 h in 60 ml of 6 M urea containing 0.1% SDS. The gel suspension was centrifuged at 10,000 x g for 30 min to remove the gel fragments, and the supernatant was saved. The elution procedure was repeated, and then the two supernatants were combined and concentrated to 15 to 20 ml by ultrafiltration on an Amicon YM5 membrane (Amicon Corporation, Danvers, Mass.). To reduce the amount of Tris, which can interfere with the removal of SDS (8), the sample was washed twice by adding 50 ml of 6 M urea and concentrated as before. The purity of this sample was determined by analytical SDS-PAGE of a fraction which had been further concentrated on an Amicon Centricon-10 unit. Removal of SDS. To remove SDS, the proteins were chromatographed on a 26- by 1.5-cm column of AG 11A8 ion retardation resin (Bio-Rad) by the method of Kapp and Vinogradov (8), except that 6 M urea was used as the eluant. The column was washed with 5 volumes of 1.0 M NH4Cl followed by 20 volumes of distilled water and 1 volume of 6 M urea before use. The protein-containing fractions in the column eluate were pooled and concentrated to 300,ug/ml by ultrafiltration on a YM5 membrane. Renaturation. The purified proteins were dialyzed in Spectrapor dialysis tubing, 12,000 to 14,000 molecular weight cutoff (Spectrum Medical Industries Inc., Los Angeles, Calif.), for 15 h at 4 C against 10 mm sodium acetate buffer (ph 4.5). The proteins were dialyzed again for 3 h against 10 mm sodium-potassium phosphate buffer (ph 6.0), containing 0.02% NaN3, to adjust the ph to that used in the hemagglutination assay. Then the preparations were examined for the presence of fimbriae by electron microscopy and tested for hemagglutination. For the latter test, the assay described below was scaled down 10-fold, and the agglutination was rated visually because the amount of purified fimbrial protein was a limiting factor. To examine the effect of ph on renaturation, 10 mm glycinehydrochloride buffer at ph 3.0; 10 mm sodium acetate buffers at ph 3.5, 4.0, 4.5, and 5.0; and sodium phosphate buffers at ph 6.0 and 7.0 were used in the first dialysis step. Precipitation by cations. To 1 ml of a protein preparation, which had been obtained by precipitation from the crude extract with acetic acid, redissolved by the addition of NaOH, and adjusted to ph 6.0 as described above, was added 0.5 ml of the appropriate salt solution at various concentrations. Ca2+, Mg2+, Na+, and K+ were added as the chloride salts, and Zn2+ was added as ZnSO4. After incubation of these at 40 C for 15 min, the samples were centrifuged for 3 min at 1,700 x g. The supernatants were dialyzed against 10 mm sodium-potassium phosphate buffer (ph 6.0), containing 0.02% NaN3, and then assayed for protein and hemagglutinating activity. The amounts of protein and hemagglutinin that precipitated were calculated from the reduction in protein concentration and hemagglutinating activity of the supernatants as compared with an uncentrifuged control to which 0.5 ml of distilled water had been added instead of salt solution. Hemagglutination assay. A quantitative assay based on the method of Liener (15) was used. Serial twofold dilutions of the agglutinin were made in 10 mm sodium-potassium phosphate buffer (ph 6.0), containing 0.02% NaN3, to a final volume of 2 ml each in 11- by 100-mm test tubes. A suspension of glutaraldehyde-treated human group A erythrocytes (Sigma) at approximately 0.4% was prepared in the same buffer containing 0.9% NaCl, such that a twofold dilution of the suspension had an optical density at 620 nm of 0.5 in a Bausch and Lomb Spectronic 20 colorimeter. Two milliliters of this suspension was added to each dilution of the agglutinin, the mixture was left undisturbed at room temperature for 2 h in an exactly vertical position, and then the optical density at 620 nm was measured. One hemagglutination unit is defined as the amount of agglutinin required to cause a 50% decrease in absorbance in 2 h, and the specific hemagglutinating activity was calculated from the equation described by Liener (15). Electron microscopy. Fimbriae preparations were applied to Formvar-coated copper grids and stained with 1% (wt/vol) phosphotungstic acid adjusted with KOH to ph 6.5. The grids were examined in a JEM 7 electron microscope and photographed at a magnification of 17,800. The diameters of native and renatured fimbriae were calculated from measurements of projections, enlarged 10 times, of the photographic plates. Amino acid analysis. Protein hydrolysis was performed under vacuum in 6 N HCI and 0.2% (vol/vol) phenol for 20 h (17). The concentrations of amino acids in the hydrolysate were determined with a Durrum amino acid analyzer (model D-500) at the Amino Acid Analysis Facility, Biophysics Laboratory, University of Wisconsin-Madison. Analytical methods. Protein was determined by the method of Lowry et al. (16) with ovalbumin as the standard. Carbohydrate was assayed by the phenol-sulfuric acid method (2) and expressed as glucose equivalents. The method of Karkhanis et al. (9) was used to measure 2-keto-3- deoxyoctonate. RESULTS Purification of the fimbrial hemagglutinin. The crude, distilled water extract of P. solanacearum contained many fimbriae, about 6 nm in diameter (Fig. 1A), and showed strong hemagglutinating activity. In the hemagglutination assay, stronger agglutination occurred when serial dilutions of the agglutinin were prepared in a buffer of low ionic strength (10 mm sodium-potassium phosphate buffer containing 0.02% NaN3) instead of phosphate-buffered saline (containing 0.9% NaCl), which is normally used in such assays, since the agglutinin precipitated from solution at the higher salt concentration. Although the erythrocyte suspension, which was then added to dilutions of the agglutinin, contained phosphate-buffered saline to stabilize the suspension, a similar effect of salt on the agglutination was not apparent. This suggests that the agglutinin binds to the erythrocyte surface before precipitation occurs. Analysis of the crude fimbrial preparation by SDS-PAGE indicated the presence of one major protein band with a molecular weight of 9,500 and of numerous minor bands (Fig. 2). Most of the minor proteins appeared to be components of a high-molecular-weight complex originally associated with the 9.5-kilodalton (9.5K) protein in the crude extract. This complex could not be separated by column

3 VOL. 50, 1985 REASSEMBLY OF FIMBRIAL HEMAGGLUTININ 607 FIG. 1. Electron micrographs of fimbriae from P. solanacearum. (A) Fimbriae in the crude extract. (B) Fimbriae obtained by renaturation of the 9.5K protein subunit isolated by preparative SDS-PAGE. Bar, 0.2,um. isoelectricfocusing or density gradient centrifugation in CsCl or sucrose, and eluted at the void volume when the crude extract was chromatographed on Sepharose 4B (exclusion limit 2 x 107) in the presence of 6 M urea (data not shown). For these reasons we used preparative SDS-PAGE, which combines strong dissociating conditions and high resolving power, to isolate the 9.5K protein in the hope that it could subsequently be renatured and identified. Before SDS- PAGE, most of the carbohydrate was removed from the crude extract by the two protein precipitation steps described below. If these steps were not included, the 9.5K protein contained up to 20% carbohydrate when isolated from the gel. This carbohydrate contained 2-keto-3- deoxyoctonate, indicating contamination of the protein with lipopolysaccharide. When the ph of the crude extract was lowered to 3.0 by adding acetic acid (1% [vol/vol]) and the resulting precipitate was removed by centrifugation, neither fimbriae nor hemagglutinating activity were detected in the supernatant but were recovered in the resolubilized pellet. Most of the Lowry-positive material removed from the crude extract at this step was of low molecular weight (not retained by dialysis tubing with a molecular weight cutoff of 12,000), and SDS-PAGE indicated that very few polypeptides had been removed (Fig. 2). When CaCl2 (20 mm) was added to the A B C D E F FIG. 2. SDS-PAGE analysis of P. solanacearum fimbrial preparations at various stages of purification. Lane A, crude extract; lane B, fraction precipitated by acetic acid (1% [vol/vol]); lane C, fraction precipitated by 20 mm CaCl2 after resolubilizing the acid precipitate; lanes D and E, protein purified by preparative SDS-PAGE (lane D, 100,ug; lane E, 30,ug); lane F, standard proteins, ovalbumin (43,000), a-chymotrypsinogen (25,700), P-lactoglobulin (18,400), and bovine trypsin inhibitor (6,200). Lysozyme and cytochrome c run as a single band (13,300) unless a longer gel is run. Molecular weights (x 103) of the standards are shown to the right of lane F.

4 608 YOUNG ET AL. TABLE 1. Precipitation by various cations of hemagglutinating activity and protein in a partially purifieda preparation of P. solanacearum fimbriae Cation Concn (mm) Precipitation Protein Hemagglutinin None 0 0 Ca Zn Mg Na K a The crude extract was treated with acetic acid and centrifuged, and the pellet was redissolved and then adjusted to ph 6 as described in the text. resolubilized pellet at ph 6.0, both fimbriae and hemagglutinating activity precipitated out and they were not detected in the supernatant. Mg2+ and Zn2+ were similar to Ca2+ in their ability to precipitate both protein and hemagglutinating activity from partially purified preparations, whereas the monovalent ions Na+ and K+ were much less effective (Table 1). Analysis by SDS-PAGE of the protein fraction recovered after precipitation by 20 mm CaCl2 showed that most of the proteins in the crude extract were still present (Fig. 2), again indicating the presence of a multiprotein complex in the crude extract. Results of preliminary experiments showed that labeling of the fraction precipitated by CaCl2 with fluorescamine did not affect the mobility of the 9.5K band on SDS-PAGE. Thus, labeling of part of the sample before preparative SDS-PAGE allowed accurate excision of the 9.5K band under UV light after electrophoresis. Subsequent analysis of this isolated band by SDS-PAGE showed a single protein (Fig. 2) which did not stain for carbohydrate. This protein had high hemagglutinating activity (Table 2) and contained a TABLE 2. Recoveries of protein, carbohydrate, and hemagglutinating activity during purification of P. solanacearum fimbriae *Protein Carbohydrate Hemagglutination Purification stage (mg) (mg) (HU/mg of (mg) (mg) ~~~protein)' Crude extract Acetic acid precipitation CaCl2 precipitation SDS-PAGE AG 11A8, renaturationb 3.0' a Preparations were dialyzed against 10 mm sodium-potassium phosphate buffer (ph 6.0) before assaying for hemagglutination. HU, Hemagglutination unit. b Protein-containing fractions eluted from the AG 11A8 column were renatured by dialysis for 15 h against 10 mm sodium acetate buffer (ph 4.5). C The final yield of purified fimbriae was 0.35 mg/g (wet wt) of bacteria. APPL. ENVIRON. MICROBIOL. TABLE 3. Amino acid composition of P. solanacearum fimbriae Amino acid Mol (%) Residues per subunita Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cystine (half) 0.48 lb Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine a The molecular weight of the fimbrial protein subunit was estimated to be 9,500 by analytical SDS-PAGE. bsince cystine recoveries are variable, this value should be considered only a rough estimate. relatively high proportion (41.8%) of hydrophobic amino acids (proline, glycine, alanine, valine, methionine, isoleucine, leucine, and phenylalanine) (Table 3). Renaturation. For renaturation of the purified 9.5K protein, it was necessary to remove the SDS by chromatography on AG 11A8 in 6 M urea and then to remove the urea by dialysis for 15 h against 10 mm sodium acetate at ph 4.5. This yielded reassembled fimbriae which had high hemagglutinating activity and had the same diameter as those in the crude extract but were much longer (Fig. 1B). These reassembled fimbriae tended to associate lengthwise into strands of varying thickness. The slightly granular appearance of fimbriae in the crude extract, which appears to result from their association with vesicular material, was not seen in the purified and renatured preparations. Based on the amount of hemagglutinating activity recovered and the relative concentrations of fimbriae determined by electron microscopy, renaturation occurred on dialysis against buffers (10 mm) in the ph range of 3 to 5, with the optimum being about ph 4.5. Renaturation did not occur at ph values of 6.0 or 7.0. Renaturation at ph 4.5 was unaffected by the inclusion of CaCl2, MgCl2, or EDTA (each at 1 mm) in the dialysis buffer. The recovery of hemagglutinating activity on protein renaturation varied considerably between preparations. Typically, however, the specific hemagglutinating activity of the renatured protein was in the same range as that of the crude extract. During renaturation, partial precipitation of the reassembled fimbriae occurred, and this became more pronounced when preparations were dialyzed beyond 15 h. This increased precipitation was accompanied by a reduction in hemagglutinating activity. Attempts to resolubilize the precipitated protein by raising the ph or by dialyzing against 6 M urea were unsuccessful. DISCUSSION Conventional techniques were unsuitable for purification of Bi fimbriae because of their tendency to bind tenaciously to other proteins and to lipopolysaccharides. Similar difficulties have been encountered in attempts to purify Escherichia coli type I fimbriae (12), presumably the result of

5 VOL. 50, 1985 strong hydrophobic or charge-charge interactions. The work described here demonstrates that preparative SDS-PAGE is an effective means of purifying Bi fimbriae. The method may be applicable to other types of fimbriae. Although several oligomeric enzymes have been successfully renatured, with recovery of their original quaternary structure after denaturation with SDS (23), to our knowledge this has not previously been reported for fimbriae. Previous attempts to reassemble fimbriae after their dissociation into protein subunits have met with limited success. When E. coli type I fimbriae were depolymerized by saturated guanidine hydrochloride at 37 C and the detergent was removed by dialysis in the presence of MgCl2, the subunits reassembled into structures resembling very short fragments of intact fimbriae (4). Subunits obtained by dissociating Pseudomonas aeruginosa fimbriae with octyl glucoside also were found to reassemble into short rods on dialysis (22), but these structures had a larger diameter than native fimbriae and did not show the same biological properties. In contrast with these reports, the renaturation and reassembly of subunits of P. solanacearum fimbriae by our method yields fibers that appear to have the same structural and binding properties as native fimbriae. This is based on the diameter and ability to cause hemagglutination of the reassembled fimbriae. In contrast to E. coli type I fimbriae (4), the reassembly of P. solanacearum fimbriae showed no requirement for divalent cations. It has been suggested that the proper in vivo assembly of fimbriae from subunits might require a proton motive force (22), analogous to that required for assembly of the filamentous phage M13 (18). Our results, however, indicate that the assembly of P. solanacearum fimbriae can occur in vitro in the absence of an energy source. An alternative method we have used to purify Bi fimbriae involves repeated sonication of crude extracts in the presence of sodium deoxycholate, which removes contaminants bound to the fimbriae (20a). Although this procedure is useful for purifying large amounts of fimbriae, the resulting preparation still contains an appreciable amount of carbohydrate. Also, we have found that extensive sonication fragments Bi fimbriae and destroys their ability to agglutinate erythrocytes. The amino acid composition of Bi fimbriae is very similar to that of other fimbriae (7) with respect to the high levels of aspartic and glutamic acids and the relatively high proportion of hydrophobic amino acids. The precipitation of P. solanacearum fimbriae at low ph is also consistent with an acidic protein. It has been shown that the Bi strain is agglutinated by divalent cations at low concentrations (Ca2+ or Zn2+ at 2 mm), whereas Na+ and K+ cause agglutination only at concentrations.100 mm (Y. Mino and L. Sequeira, unpublished data). This agglutination apparently is mediated by fimbriae and may be related to differences in the behavior of virulent and avirulent strains of the bacterium in planta. Strain Bi, for example, is readily immobilized on tobacco cell walls, whereas the virulent, parental strain K60 which produces very few fimbriae, is not immobilized (20; 20a). The discovery that divalent cations precipitate Bi fimbriae in crude or partially purified preparations, whereas monovalent cations are much less effective, strongly implicates fimbriae or a fimbriae-associated molecule in ion-mediated agglutination. The latter possibility must be considered for we have been unable to demonstrate precipitation of the purified, renatured fimbriae by 20 mm CaCl2 (data not shown). The possible involvement of fimbrial-bound lipopolysaccharide REASSEMBLY OF FIMBRIAL HEMAGGLUTININ 609 in ion-mediated agglutination is worth considering since MgCl2 (25 mm) is known to precipitate certain rough lipopolysaccharides (19). The interaction of Bi fimbriae with the erythrocyte surface and the plant cell wall has been under investigation and will be discussed in a later report. ACKNOWLEDGMENTS This work was supported by project 1474 from the College of Agricultural and Life Sciences, University of Wisconsin-Madison and by grants from the National Science Foundation and The Rockefeller Foundation. The technical assistance of G. Gaard with electron microscopy and S. Vicen with photography is gratefully acknowledged. LITERATURE CITED 1. Anderson, B. L., R. W. Berry, and A. Telser A sodium dodecyl sulfate-polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000. Anal. Biochem. 132: Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: Duvick, J. P., and L. Sequeira Interaction of Pseudomonas solanacearum lipopolysaccharide and extracellular polysaccharide with agglutinin from potato tubers. Appl. Environ. Microbiol. 48: Eshdat, Y., F. J. Silverblatt, and N. Sharon Dissociation and reassembly of Escherichia coli Type I pili. J. Bacteriol. 148: Fairbanks, G., T. L. Stack, and D. F. H. Wallach Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10: Fuerst, J. A., and A. C. Hayward Surface appendages similar to fimbriae (pili) on Pseudomonas species. J. Gen. Microbiol. 58: Jones, G. W., and R. E. Isaacson Proteinaceous bacterial adhesins and their receptors. Crit. Rev. Microbiol. 10: Kapp, 0. H., and S. N. Vinogradov Removal of sodium dodecyl sulfate from proteins. Anal. Biochem. 91: Karkhanis, Y. D., J. Y. Zeltner, J. J. Jackson, and D. J. Carlo A new and improved microassay to determine 2-keto-3- deoxyoctonate in lipopolysaccharide of gram-negative bacteria. Anal. Biochem. 85: Kelman, A The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44: Kelman, A., and J. Hruschka The role of motility and aerotaxis in the selective increase of avirulent bacteria in still broth cultures of Pseudomonas solanacearum. J. Gen. Microbiol. 76: Korhonen, T. K., E. Nurmiaho, H. Ranta, and C. Svanborg Eden New method for isolation of immunologically pure pili from Esc herichia coli. Infect. Immun. 27: Leach, J. E., M. A. Cantrell, and L. Sequeira Hydroxyproline-rich bacterial agglutinin from potato. Extraction, purification, and characterization. Plant Physiol. 70: Leach, J. E., M. A. Cantrell, and L. Sequeira A hydroxyproline-rich bacterial agglutinin from potato: its localization by immunofluorescence. Physiol. Plant Pathol. 21: Liener, I. E The photometric determination of the hemagglutinating activity of soyin and crude soybean extracts. Arch. Biochem. Biophys. 54: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Moore, S The precision and sensitivity of amino acid analysis, p In J. Meienhofer (ed.), Chemistry and

6 610 YOUNG ET AL. APPL. ENVIRON. MICROBIOL. Biology of Peptides: Proceedings of the Third American Peptide Symposium. Ann Arbor Science, Ann Arbor, Mich. 18. Ng, Y. C., and A. K. Dunker Effects of energy uncouplers on fd phage assembly, p In Proceedings of the VIIth Biennial Conference on Bacteriophage Assembly. Alan R. Liss Publishers, New York. 19. Osborn, M. J Preparation of lipopolysaccharide from mutant strains of Salmonella. Methods Enzymol. 8: Sequeira, L., G. Gaard, and G. A. DeZoeten Interaction of bacteria and host cell walls: its relation to mechanisms of induced resistance. Physiol. Plant Pathol. 10: a.Stemmer, W. P. C., and L. Sequeira Possible role of fimbriae in attachment of plant-associated bacteria to plant cell walls, p In A. A. Szalay and R. P. Legocky (ed.), Proceedings of the 2nd International Symposium on the Molecular Genetics of the Bacteria-Plant Interaction. Media Services, Cornell University, Ithaca, N.Y. 21. Udenfriend, S., S. Stein, P. Bohlen, W. Dairmann, W. Leimgruber, and M. Weigele Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178: Watts, T. H., D. G. Scraba, and W. Paranchych Formation of 9-nm filaments from pilin monomers obtained by octylglucoside dissociation of Pseudomonas aeruginosa pili. J. Bacteriol. 151: Weber, K., and D. J. Kuter Reversible denaturation of enzymes by sodium dodecyl sulfate. J. Biol. Chem. 246: