A New Type of Bacterial Pilus Genetically Controlled by the Fertility Factor of E. Coli K 12 and Its Role in Chromosome Transfer.

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 Fstrain. 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 Fstrains, 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