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Ff Coliphages: Structural and Functional Relationships MICROBIOLOGICAL REVIEWS, Dec. 1986, p. 401-427 Vol. 50, No. 4 0146-0749/86/040401-27$02.00/0 Copyright © 1986, American Society for Microbiology Ff Coliphages: Structural and Functional Relationships IHAB RASCHED* AND ERIKA OBERER Faculty for Biology, University of Konstanz, D-7750 Konstanz, Federal Republic of Germany INTRODUCTION .................................... 401 MORPHOLOGY AND STRUCTURE .................................... 401 Comparison with Other Filamentous Viruses .................................... 403 GENE PRODUCTS .................................... 403 Survey .................................... 403 Replication Proteins .................................... 404 Gene 2 protein .................................... 404 Gene 5 protein .................................... 405 Coat Proteins .................................... 406 Major coat protein .................................... 406 Minor coat proteins .................................... 408 PHAGE PROPAGATION (PROLIFERATION) .................................... 409 Adsorption and Penetration (Infection) .................................... 410 Progeny Production (Assembly) .................................... 410 GENETICS .................................... 411 The Genome .................................... 411 DNA Sequencing and Structure Investigations .................................... 412 414 DNA Replication .................................... 414 Stage 1 .................................... 415 Stage 2 .................................... 416 Stage 3 .................................... Transcription and Translation .................................... 416 418 Cloning Vectors .................................... CONCLUDING REMARKS .................................... 418 ACKNOWLEDGMENTS .................................... 419 LITERATURE CITED .................................... 419 INTRODUCTION ally make no distinction between research done with one or the other phage. Filamentous bacteriophages are a group of related viruses which only infect gram-negative bacteria and specifically MORPHOLOGY AND STRUCTURE adsorb to the tip of pili.They are long and thin particles, threadlike, with no organization into head or spikes. Their The general appearance of bacteriophage fd (fl, M13) is genetic material consists of a single-stranded, closed circular that of a flexible filament, about 900 nm long and 6 to 10 nm deoxyribonucleic acid (DNA) molecule. For an excellent thick, with a molecular weight of 12 x 106 (17, 120, 186, 220). review classifying the several types of bacteriophages and Its coat consists of 2,700 to 3,000 copies of a major protein, describing them in detail, published in 1969, see Marvin and the product of gene 8 (g8p) (220, 247), and four minor Hohn (187). components, the products of genes 3, 6, 7, and 9 (g3p, g6p, In this article, we will deal exclusively with the most g7p, g9p), present in ca. five copies each. It contains no lipid thoroughly investigated Ff phages, fd, fl, and M13, which or carbohydrate. The coat envelopes one molecule of DNA infect Escherichia coli. Ff denominates those filamentous that extends the total length of the virus and constitutes ca. phages which require F pili as the host receptor. Accord- 12% of its weight (16, 220). The DNA is single stranded, ingly, they are male specific (androphage) and would not use closed circular, with no Watson-Crick base pairing except other pili also found on the surface of certain E. coli cells. fl for a few specific regions (120, 186). It is about 6,400 was first isolated at Rockefeller University in 1960 and nucleotides long, codes for 10 proteins, and has a molecular described by Loeb (171) and Zinder et al. (356); M13 was weight of 2 x 106 (64, 220). discovered in Munich (121) and fd was discovered in The phage filamentous structure is highly sensitive to Heidelberg (185, 186). They resemble each other so closely mechanical shearing and ultrasonic treatment. Also, in con- that it is legitimate to consider them as slight mutations of trast to X-like phages, it is sensitive to detergents and organic basically the same phage. Except for a small number of base solvents, especially chloroform. On the other hand, the virus changes their DNA sequences are identical. The same holds is rather resistant to proteolytic and nucleolytic digestion, true for the composition of their coats as visualized in with the sole exception of subtilisin, which partially digests sodium dodecyl sulfate (SDS)-polyacrylamide gel electro- one of the minor coat proteins, the gene 3 product (187). phoresis. Throughout this review, we will therefore gener- By using X-ray diffraction techniques, the molecular ar- chitecture of Ff has been extensively investigated, and the following structural features have been discussed (9, 188, * Corresponding author. 190): the filament is a hollow protein cylinder with an outer 401 402 RASCHED AND OBERER MICROBIOL. REV. diameter of 6 nm and an inner diameter of 2 nm. Other data acidic amino acids are accumulated in the N-terminal part concerning the inner diameter (4 nm) have also been re- which is exposed on the virus surface and determines the ported (320). The DNA molecule is embedded in the cylinder electrophoretic mobility of whole phage in gels (23); near the along its longitudinal axis. Two alternative models for the C terminus basic amino acids predominate and supposedly coat protein arrangement around the DNA have been dis- interact with the negatively charged DNA phosphate back- cussed (188-190, 320). The so-called one-strand model pro- bone (5); and finally, hydrophobic residues are concentrated poses that the two antiparallel chains of the DNA molecule in the central section of the sequence (336). This hydropho- extend along the central axial hole of the cylinder. In the bic "core" is in contact with the corresponding domains of alternative "two-strand model" (105), the "up" and neighboring subunits, leading to the tight packing of mole- "down" strands of the circular genome are separately cules in the coat seen in X-ray and nuclear magnetic reso- wrapped with coat protein and then twisted around each nance analyses. The detailed structure of the major coat other. protein will be dealt with in a later section. The compiled data, however, definitely support the "one- Chaotropic agents such as guanidinium hydrochloride, strand model" (320). The DNA packaged in native Ff virus SDS, or cholate dissociate the phage coat into the major is folded back on itself with no protein inside; the molecule protein monomers. This dissociation is highly cooperative is dense due to the extensively stacked nucleotide bases and (all-or-none mechanism) (134). The same is true for alkali- has a fixed orientation (65, 282): a major hairpin structure induced disassembly (264), whereas heat inactivation of the (hairpin A; see section, "Genetics"), which is located near phage induces intermediate states (262, 263). the origins of replication, is invariably found at one end of One common structural feature of filamentous phages is the phage filament, no matter how long the DNA is (135, the existence of still another complex of viral DNA and viral 327). The DNA forms a helix with a radius of 0.85 nm and a protein during their "life cycle." The protein in this case is pitch of about 2.67 nm; the rise per nucleotide is 0.272 nm the product of gene 5, which has a decisive role in the (9). synthesis of progeny single-stranded DNA and may be The major coat protein subunits form a helix of their own. regarded as scaffolding protein in the early stage of phage On the basis of their X-ray diffraction pattern, filamentous assembly (see below). bacteriophages can be separated into two classes, I and II. In contrast to the major coat protein, which is uniformly The class II pattern (190), displayed for example by phage distributed along the phage envelope, the minor coat pro- Pfl (infecting Pseudomonas aeruginosa) or XF (infecting teins are located exclusively at the ends of the particle. Xanthomonas oryzae), can be interpreted as a simple one- Unfortunately, X-ray analysis of phage fibers provides no start helix of 1.5 nm pitch with 4.4 (190) or 5.4 (180) U per information about the exact arrangement of these minor turn. components. This is especially true for the products of genes fd, fl, and M13, together with phage Ifl and Ike, which all 7 and 9. They are found together at one end of the filament, infect E. coli, belong to class I (188). Their molecular while the products of genes 3 and 6 are located at the other architecture is a perturbed version of the simpler class II (96, 98, 288, 289, 345). helix, yet the two patterns are not interconvertible. The The "adsorption protein" g3p is anchored to the virus symmetry of class I particles is a five-start helix of protein coat via the C-terminal part of the polypeptide chain, subunits with an approximately twofold screw axis, 4.5 U whereas the N terminus is exposed and mediates the attach- per turn, and 1.5-nm pitch (65, 180, 188). The helix is ment of the virus to the tip of a host F pilus (6). The electron left-handed and has repeated perturbations along its axis. microscope reveals a "knob-on-stem" structure at one virus The major coat protein subunits appear as flexible, ot- end which represents the viral adsorption complex (102): the helical rods (9), "elongated in an axial direction" and sloping N-terminal part of the protein sequence is folded into the radially, "so as to overlap each other and give an arrange- "knob" and is susceptible
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