Bacterial pili a r e defined as any morphologically distinct filamentous appendage of bacterial cells other than flagella. The word pili is derived from the Latin word meaning hair o r hair-like structure. They occur very widely among gram-negative bacteria, particularly on strains freshly isolated from natural sources. Since microscopically visible pili a r e not necessary for growth o r viability under the usual laboratory conditions, piliated bacteria represent a differentiated form of the cell having special functions under special conditions. The work presented here is a brief summary of research on pili carried out between 1951 and 1965 at the Biophysics Departments of the University of Pittsburgh and the University of Geneva, the Institute for Morphologically, pili a r e too thin to be visible by ordinary light microscopy and must be visualized by electron microscopy. Although an exhaustive and systematic search for new kinds of pili has not been made, six distinct morphological types have been found and numbered in order of discovery o r named according to function (TABLE 1). Although the diameter of pili is quite constant for a given type, their length is variable even among pili on the same cell. Unlike flagella, they are straight, and some appear to be rigid.Pili a r e usually distributed evenly over the entire surface of the cell. FIGURE 1 shows Type I pili present on E. coli K12 F-, FIGURE 2 shows Type IV pili and flagella on Proteus; FIGURE 3 shows Type 111 pili, Type IV pili and flagella on Proteus; and FIGURE 4 shows Type I, Type V and F pili on E . coli K12 HfrC. The F pili a r e coveredwiththe RNA male phage M12 for which they are the receptor. The Type IV pili have a helical surface fine structure of sufficiently long pitch distance to be visible in the micrograph. Only Type I pili and F pili have been studied in detail.Type I pili may be isolated, concentrated and purified by several techniques.5 The most convenient is to remove them from the cells by two minutes of mechanical agitation in a high speed mixer and separate them from the depiliated cells by low speed centrifugation. An isoelectric precipitation at PH 3.92 serves to aggregate the pili in the supernatant liquid along with some of the impurities, and they may be concentrated by low speed centrifugation. The resuspended and neutralized preparation is then made 0.1 M with MgC12 in the cold. This specifically aggregates Type I pili, and strong streaming birefringence appears. The crystalline pili aggregates are removed by low speed centrifugation and resuspended in distilled water. Two more cycles of magnesium precipitation yield a pili suspension which is pure by the criteria of UV spectroscopy, electron microscopy, chemical analysis, crystallinity, *This paper, illustrated with slides, was the second of two papers to be presented at 1003 a meeting of the Division.

No abstract

The type-1 pilus of Escherichia coli is the prototype of this class of hair-like, multimeric adhesive organelles. This pilus mediates adherence to mannose-containing receptors on mucosal epithelia and other cells. The type-1 pilus, in one of several serological variants, is expressed by nearly all E. coli strains, and its promotion of colonization by pathogenic bacteria and the protective effects of purified pilus vaccines suggest that it is important as a bacterial virulence factor. Both the adhesive function and the serological variation of the type-1 pilus have been attributed to the thousand or so pilin protein monomers making up the pilus rods. This idea has been contradicted by our earlier observations on an E. coli strain expressing adhesion-defective pili. More recent genetic evidence also indicates that auxiliary pilus proteins are required for adhesive function. We report here the identification of three previously undetected integral minor proteins on the type-1 pilus, and show that one of them is the receptor-binding adhesin. This protein is antigenically conserved among strains with different pilin serotypes and is located at the pilus tip.

Pili comprise several types of morphologically similar thin appendages growing out from the surface of gram-negative bacteria.'-3 Type I pili are composed of protein subunits of molecular weight 17,000 polymerized into rigid right-handed helices of diameter 70 A and pitch 24 A, having an axial hole 20-25 A in diameter.4 Other types of pili with different external diameters exist2 but their composition and fine structure is unknown.It had been considered previously that pili might be involved in the fertility of male bacteria.3 This speculation was based on the frequent occurrence of pili on male E. coli K 12 strains and on the plausibility of chromosomal transfer being mediated by a rod-like structure which could actively traverse the cell membrane and wall. However, no correlation of piliation with maleness was found. Many female strains were richly piliated and some cultures of male strains contained only a few sparsely piliated cells. The hypothesis could not be disproved, however, since none of the many male strains subsequently examined were ever completely nonpiliated. Although the majority of cells in a culture may have had no pili, at least a few cells could always be found with a few attached pili.A new method of approach to chis problem has been provided by the recent electron microscopic observations of Crawford and Gesteland5 who noted that a male-specific bacteriophage, R-17, adsorbed to pili of an Hfr and an F+ strain of E. coli but not to pili of an F-strain.We have investigated the adsorption of another male-specific bacteriophage, M 12, isolated by P. H. Hofschneider.8 Our studies revealed that M 12 adsorbs to some of the pili present on male bacteria and that phage adsorption can be used to distinguish them in electron micrographs from other types of pili occurring on the same cell. It is possible to demonstrate that these "F pili" are genetically controlled by the fertility factor of E. coli K 12.Materials and Methods.-Phage: M 12 phage, a small (about 270 A diameter), spherical, RNA-containing phage infecting Hfr and F+ strains but not F-strains, was obtained from Dr. P. H. Hofschneider.Electron microscopy: Bacteria were grown in tryptone yeast extract calcium broth (10 gm tryptone, 5 gin yeast extract, 5 gm NaCl, 0.75 gm CaCl2 2H20 per liter of water, the CaC12 sterilized separately). Overnight, unshaken, unaerated cultures were diluted 1:10 into fresh medium and grown for 3-4 hr under the same conditions until the bacterial density was about 5.108 cells per ml. M 12 phage was added at multiplicities from 5 to 100 and the mixture incubated at 370C. After 10 min, the mixture was rapidly chilled in an ice bath and prepared for the electron microscope by the collodion agar filtration method of Kellenberger.7 Tests for phage susceptibility: (A) Plaque formation: Standard phage plaque methods using tryptone yeast extract calcium soft agar (0.7%) and bottom agar 776

ABSTRACr A method is described for the retrieval of streptavidin from the culture broth of Streptomyces avidinii. The key step in this procedure is the adsorption of streptavidin from culture concentrates to an affinity column in which iminobiotin is attached to AH-Sepharose 4B. This column binds streptavidin at pH 11 and releases the protein at pH 4. The recovery of streptavidin is practically quantitative. The pH dependence of the iminobiotin-avidin affinity, discovered by Green [Green, N. M. (1I) Biochem. 1. 101, 774-7791, has thus found practical application. The streptavidin bound 4.07 1 0.02 mol of [14C]biotin per mol and was essentially homogeneous as judged by disc and slab gel electrophoresis. Streptavidin was extensivey succinoylated without loss of biotin-binding capacity. The observations that 12SI-labeled streptavidin and I15I-labeled succinoylstreptavidin are retained by iminobiotin-AH-Sepharose 4B columns at pH 7.5 and are eluted at pH 4.0 provides a convenient purification method for these iodinated proteins. The technique employed for the retrieval of streptavidin is generally applicable to the isolation of iminobiotinylated molecules.In recent communications (1, 2) we proposed a scheme for the labeling of peptide and protein hormones involving the noncovalent attachment of 125I-labeled avidin of high specific radioactivity to biotinylated hormones. To test this idea a procedure was developed for labeling avidin with 1251 to high specific radioactivity st2 mCi/nmol; 1 Ci = 3.7 X 1010 bec.-querels. The labeled pHPP-avidint bound avidly to rat liver plasma membranes and was not displaceable by unlabeled avidin. Clearly 125I-pHPP-avidin was not a suitable label for biotinylinsulin. Because we suspected that the basic nature of avidin (isoelectric point 10.5) could contribute to its affinity for the essentially negatively charged plasma membranes, we subjected pHPP-avidin to extensive succionylation with succinic anhydride. This modification did not significantly alter the binding affinity for biotin, but the 125I-labeled Suc-pHPPavidint bound less firmly to the plasma membranes and was displaceable by unlabeled Suc-pHPP-avidin. With The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99', or type 1 pilus' strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99' strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili.

Pregnant gilts were vaccinated with purified strain 987 pili (987P), strain K99 pili, or a saline-formaldehyde control. Suckling pigs born to vaccinated gilts were allowed to consume colostrum and were then challenged intragastrically with one of three enterotoxigenic Escherichia coli strains: 987 (09:K103, 987P:NM), 74-5208 (020:K101, 987P:NM), or 431 (0101:K30, 99:NM). In litters where the dam was vaccinated with the same pilus as that possessed by the challenge organism, the incidence and duration of diarrhea and the degree of intestinal colonization (either duration or extent) were less than those of the other vaccine groups. Surviving pigs in the homologous vaccine groups also had better weight gains than pigs in the other vaccine groups. These experiments extend and confirm previous reports that vaccination of the dam with purified pili confers protection to neonatal suckling pigs against diarrheal disease caused by enterotoxigenic E. coli strains that possess the same pili. Protection did not extend to enterotoxigenic strains possessing different pili.

No abstract