Filamentous Bacteriophages: Biology and Applications

Abstract

Filamentous bacteriophages contain a circular single‐stranded deoxyribonucleic acid (ssDNA) genome packaged into long filaments. These phages do not reproduce by lysing bacteria; instead, they are secreted into the environment without killing the host. Well‐studied Escherichia coli K12‐infecting Ff phages (f1, fd or M13) always replicate episomally; however a growing number of ‘lysogenic’ or chromosomally integrated filamentous phages of Gram‐negative bacteria are being discovered. The ‘lysogens’ can be induced; however phage reproduction does not require genome excision from bacterial chromosome and does not lyse the host cells. Some filamentous phages enhance the virulence of their host organisms, the most striking example being the CTXφ of Vibrio cholerae, which encodes cholera toxin. E. coli Ff phages are the workhorse of phage display technology, whose most notable ‘products’ are therapeutic recombinant antibodies. Ff are also being used in nanotechnology as templates for assembly of nanostructures, which has already led to their incorporation into a working nanobattery.

Key Concepts:

  • Filamentous bacteriophages are long filaments (6–7 nm×>500 nm) that contain a single‐stranded circular DNA genome.

  • Filamentous bacteriophages replicate via a rolling circle mechanism, one strand at a time.

  • Filamentous bacteriophages do not lyse the cells; they are released by secretion, using a dedicated filamentous phage assembly secretion system.

  • Filamentous phage secretion‐assembly requires the proton‐motive force and ATP.

  • Some filamentous phages replicate exclusively as episomes, while others also integrate their genomes into the host chromosome, forming a lysogen. Induction of the lysogen does not result in cell lysis.

  • Ff filamentous phages of E. coli (f1, M13 and fd) have been used interchangeably as vectors and helper phages in DNA sequencing, as a protein display platform in phage display technology and as a template for assembly of nanostructures in nanotechnology.

Keywords: filamentous bacteriophage; ssDNA viruses; phage display; nanotechnology; Vibrio cholerae; E. coli; Xanthomonas; Pseudomonas; bacterial surfaces; bacterial secretion

Figure 1.

E. coli cells assembling Ff phages. (a) Cells infected with a mutant phage that contains a deletion of gIII and therefore does not produce pIII, a protein required for release of phage from infected cells. Hundreds of long phage filaments emanate from the bacterium, each several times longer than wild‐type Ff. (b) Cell infected with wild‐type Ff phage. A small number of single‐length phages can be observed on the surface (From Rakonjac J. PhD thesis, Rockefeller University, 1998 with permission).

Figure 2.

The Ff virion. Structure, organisation and micrograph of the Ff (f1, M13 or fd) virion. (a) Model of a pVIII (major coat protein) monomer showing hydrophobic residues (green), hydrophilic residues (orange), positively charged residues (blue), and negatively charged residues (red). N and C termini of pVIII are indicated. (b) Ribbon representation of the pVIII within the filamentous phage capsid showing the shingle‐like array of helices. (c) Schematic representation of the virion indicating the positions of virion proteins. (d) Atomic force microscope image of two Ff virions, one (longer) that has encapsidated the helper phage R408 genome, and the smaller that contains a phagemid vector whose genome is smaller. (The image in (d) is reprinted with permission from Rakonjac J and Conway JF (2006) In: Rehm B (ed.) Molecular Bionanotechnology. Horizon Scientific Press, Norwich, UK, pp. 153–190.) The images of the pVIII subunits in (a) and (b) are derived from coordinates of the RCSB PDB database accession number 2cOw (Marvin et al., ).

Figure 3.

Filamentous phage infection and lysogeny. (a) A model of filamentous phage infection and DNA entry. A retractable pilus and TolQRA complex are required for infection and entry. Major coat protein (pVIII) inserts into the inner membrane as the genome (red) enters the cytoplasm. The genome is a ssDNA (+) strand. IM, inner membrane; OM, outer membrane. (b)–(d) Fate of phage DNA after entry into host cells for filamentous phages of three distinct lifestyles. (a) Episomally replicating (nonintegrative) phages, for example, Ff or Pf1. These phages replicate constitutively. The incoming ssDNA (+) strand serves as a template for synthesis of the (−) strand, resulting in a double‐stranded DNA or replicative form (RF). The RF is a template for replication of the (+) strand, initiated by the phage‐encoded replication protein pII. (c) Reversibly integrated, constitutively replicating, lysogenic phages (e.g. VGJφ). Entry is followed by episomal replication, as well as chromosomal integration. The attP site in this group of phages is a functional dif site in the RF. Integration of the RF into the host chromosome dif site is catalysed by host‐encoded XerCD recombinase. Since two functional dif sites flank the integrated phage genome, this recombination is reversible (Das et al., ). (d) Irreversibly integrated, inducible lysogens (e.g. CTXφ). These phages contain two defective dif sites in inverted orientation. These sites are not functional in the RF, however in the ssDNA (+) these two sites anneal to each other to create a functional dif sequence in the form of a forked stem–loop (attP). This reconstituted dif site recombines into the dif site located between the chromosomal DNA (grey) and a previously inserted satellite phage, for example, RS1φ or TLCφ (blue (Hassan et al., )). Replication and/or repair DNA synthesis generates the (−) strand of CTXφ, yielding a prophage (pink) flanked by the modified dif site (attL) and reconstituted dif site (attR). Expression of the negative regulator RstR (red oval, labelled ‘R’) and its binding to the regulatory sequences between two divergent promoters (PR and PA) in combination with chromosomally encoded LexA (yellow oval, labelled ‘L’), inhibits transcription of the replication protein RstA (green block arrow, labelled ‘A’), genes encoding other phage proteins (not shown) and the positive regulator RstC encoded by the satellite (blue block arrow, labelled ‘C’), while stimulating its own expression (McLeod et al., ). Key: pII (replication protein), green oval; functional dif site, black star; defective (nonfunctional) dif site, white star; origin of replication, ori; ‘o’; replicative form, RF (episomal circular dsDNA); positive and negative strand of viral ssDNA, ‘+’ and ‘−‘.

Figure 4.

Filamentous phage replication and assembly. (a) A model for filamentous phage assembly. The packaging substrate, ssDNA‐pV complex, is targeted to the assembly machinery composed of the inner membrane complex (phage‐encoded pI/pXI; a membrane‐embedded ATP‐ase) and an outer membrane channel of the secretin family. All virion proteins (pVI, pIII, pVIII, pVII and pIX) are integral membrane proteins prior to assembly into the virion. As the ssDNA traverses the inner membrane, pV is replaced by the subunits of major coat protein (pVIII). IM, inner membrane; OM, outer membrane. (b–d) Replication and formation of packaging substrate (ssDNA‐pV complex) by filamentous phages of three distinct lifestyles. (b) Episomally replicating phages (e.g. Ff or Pf1). Early in the infection, (+) strands serve as a template for replication of (−) strand, to increase the RF copy number. Later in the infection, the phage‐encoded ssDNA‐binding protein pV coats the ssDNA, forming the pV‐ssDNA complex which serves as the packaging substrate for assembly of the virion. An exposed hairpin (the packaging signal) targets the ssDNA‐pV complex to the membrane‐embedded assembly secretion machinery. (c) Constitutively replicating, reversibly integrated lysogenic phages (e.g. VGJφ). Recent study showed that the episomal replication, as well as reversible integration‐excision, appears to occur constitutively in these phages. Integration is required for maintenance of the episomal RF in a bacterial population (Das et al., ). (d) Irreversibly integrated, inducible lysogens (e.g. CTXφ). Upon induction of the SOS response by UV light or Mitomycin C, host‐encoded repressor LexA is degraded, resulting in cessation of the repressor rstR (R) transcription from the PR promoter. Consequently, the PA promoter is induced, allowing production of the positive regulator RstC (blue oval) which sequesters the remaining RstR, further inducing the PA promoter. Induction of PA promoter also results in production of the replication protein RstA (green oval), and phage‐encoded proteins required for assembly of the progeny virions (McLeod et al., ). Replication of prophage ssDNA initiates at the (+) origin of replication (ori; labelled ‘o’) located approximately 500 nucleotides downstream from the attL site, and proceeds until it reaches the ori of the satellite prophage, synthesising a new (+) strand while displacing and releasing the re‐circularised old (+) strand ssDNA. Therefore, the resulting replicon (purple) is not the same as the one that was integrated into the chromosome (red), but rather a chimera of the integrated CTXφ and the satellite. Episomal replication ensues, producing both the RF and the (+) ssDNA, which is packaged into the virion and secreted out of the cell. Phage production by lysogens is inefficient, achieving titres between 106 and 1010 per mL, whereas episomally replicating Ff phages give titres of 1012–1013 per mL. Key: Chromosome, grey; prophage, pink; satellite or defective prophage, blue; repressor RstR, red oval, labelled ‘R’; replication proteins pII or RstA, green oval; positive regulator RstC encoded by the satellite, blue oval, labelled ‘C’; functional dif site, black star; defective (nonfunctional) dif site, white star; attL and attR, left and right joints of the integrated phage/satellite genomes and the chromosome; origin of replication, ori (‘o’) in the prophage; positive and negative strand of viral DNA, ‘+’ and ‘−’.

Figure 5.

The dif (att) sites in phage genomes and V. cholerae chromosome 1. Sequences in red and blue are binding sites for subunits of XerCD recombinase, XerC and XerD, respectively. Intervening sequence (spacer) is shown in black, with the exception of nucleotides involved in stabilisation of the strand exchange, which are purple. Asterisk denotes the position of cleavage. Nucleotides in the phage sequences that match the chromosomal dif1 site (on chromosome 1 of V. cholerae genome) are underlined. CTXφ (RF) or prophage (double‐stranded form) contains two double‐stranded attP sites. The attP1 site has a spacer between the XerC‐ and XerD‐binding sequences that is too long, preventing interaction between the two subunits. The attP2 spacer lacks complementarity to dif1 sequence on the chromosome in the key residues around the cut site, preventing strand exchange and aborting recombination. In the CTXφ ssDNA (+) strand a hybrid attP that forms by annealing attP1 to attP2 is functional; attP1 provides complementarity in the spacer, whereas attP2 shortens the distance between the XerC‐ and XerD‐ binding sequences, thanks to its short spacer. Two vertical lines in the spacer (AC∥TGT) of the CTXφ attP1 denote the position of seven‐nucleotide‐long loop (GGCGCGG) that is formed by hybridisation to attP2.

Figure 6.

Phage‐templated electrode. (a) A schematic presentation of the multifunctional M13 virus with genetically engineered proteins. The pVIII (major coat protein) is modified to serve as a template for a‐FePO4 growth; pIII is engineered to have a binding affinity for single‐wall carbon nanotubes (SWNTs). (b) A schematic diagram for fabricating genetically engineered high‐power lithium‐ion battery cathodes using multifunctional viruses (pVIII–pIII system) and a photograph of the battery used to power a green LED. The biomolecular recognition and attachment to conducting SWNT networks make efficient electrical nanoscale wiring to the active nanomaterials, enabling high power performance. These hybrid materials were assembled as a positive electrode in a lithium‐ion battery using lithium metal foil as a negative electrode to power a green LED. Active cathode materials loading was 3.21 mg/cm2. The 2016 Coin Cell used is 2 cm in diameter and 1.6 mm thick. (From Lee et al., ; reprinted with permission from AAAS.)

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References

Arap W, Pasqualini R and Ruoslahti E (1998) Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279: 377–380.

Barbas CF III, Kang AS, Lerner RA and Benkovic SJ (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proceedings of the National Academy of Sciences of the USA 88: 7978–7982.

Bennett NJ, Gagic D, Sutherland‐Smith AJ and Rakonjac J (2011) Characterization of a dual‐function domain that mediates membrane insertion and excision of Ff filamentous bacteriophage. Journal of Molecular Biology 411: 972–985.

Bille E, Zahar JR, Perrin A et al. (2005) A chromosomally integrated bacteriophage in invasive meningococci. Journal of Experimental Medicine 201: 1905–1913.

Campos J, Martinez E, Izquierdo Y and Fando R (2010) VEJϕ, a novel filamentous phage of Vibrio cholerae able to transduce the cholera toxin genes. Microbiology 156: 108–115.

Cantalupo PG, Calgua B, Zhao G et al. (2011) Raw sewage harbors diverse viral populations. Molecular Biology 2: e00180‐11.

Chopin MC, Rouault A, Ehrlich SD and Gautier M (2002) Filamentous phage active on the gram‐positive bacterium Propionibacterium freudenreichii. Journal of Bacteriology 184: 2030–2033.

Chung WJ, Oh JW, Kwak K et al. (2011) Biomimetic self‐templating supramolecular structures. Nature 478: 364–368.

Click EM and Webster RE (1998) The TolQRA proteins are required for membrane insertion of the major capsid protein of the filamentous phage f1 during infection. Journal of Bacteriology 180: 1723–1728.

Das B, Bischerour J and Barre FX (2011) VGJphi integration and excision mechanisms contribute to the genetic diversity of Vibrio cholerae epidemic strains. Proceedings of the National Academy of Sciences of the USA 108: 2516–2521.

Davis BM, Lawson EH, Sandkvist M et al. (2000) Convergence of the secretory pathways for cholera toxin and the filamentous phage, CTXϕ. Science 288: 333–335.

Day LA (2011) Family Inoviridae. In: King AMQ, Adams MJ, Carstens EB and Lefkowitz EJ (eds) Virus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses, pp. 375–384. San Diego: Elsevier Academic Press.

Day LA, Marzec CJ, Reisberg SA and Casadevall A (1988) DNA packing in filamentous bacteriophages. Annual Review of Biophysics and Biophysical Chemistry 17: 509–539.

Derbise A, Chenal‐Francisque V and Pouillot F (2007) A horizontally acquired filamentous phage contributes to the pathogenicity of the plague Bacillus. Molecular Microbiology 63: 1145–1157.

Di Niro R, Sulic AM, Mignone F et al. (2010) Rapid interactome profiling by massive sequencing. Nucleic Acids Research 38: e110.

Endemann H and Model P (1995) Location of filamentous phage minor coat proteins in phage and in infected cells. Journal of Molecular Biology 250: 496–506.

Feng JN, Russel M and Model P (1997) A permeabilized cell system that assembles filamentous bacteriophage. Proceedings of the National Academy of Sciences of the USA 94: 4068–4073.

Haigh NG and Webster RE (1999) The pI and pXI assembly proteins serve separate and essential roles in filamentous phage assembly. Journal of Molecular Biology 293: 1017–1027.

Hassan F, Kamruzzaman M, Mekalanos JJ and Faruque SM (2010) Satellite phage TLCϕ enables toxigenic conversion by CTX phage through dif site alteration. Nature 467: 982–985.

Heilpern AJ and Waldor MK (2000) CTXϕ infection of Vibrio cholerae requires the tolQRA gene products. Journal of Bacteriology 182: 1739–1747.

Higashitani N, Higashitani A, Guan ZW and Horiuchi K (1996) Recognition mechanisms of the minus‐strand origin of phage f1 by E. coli RNA polymerase. Genes Cells 1: 829–841.

Holland SJ, Sanz C and Perham RN (2006) Identification and specificity of pilus adsorption proteins of filamentous bacteriophages infecting Pseudomonas aeruginosa. Virology 345: 540–548.

Huang Y, Chiang CY, Lee SK et al. (2005) Programmable assembly of nanoarchitectures using genetically engineered viruses. Nano letters 5: 1429–1434.

Kawai M, Uchiyama I and Kobayashi I (2005) Genome comparison in silico in Neisseria suggests integration of filamentous bacteriophages by their own transposase. DNA Res 12: 389–401.

Korotkov KV, Gonen T and Hol WG (2011) Secretins: dynamic channels for protein transport across membranes. Trends Biochem Sci 36: 433–443.

Kuo TT, Lin YH, Huang CM et al. (1987) The lysogenic cycle of the filamentous phage Cflt from Xanthomonas campestris pv. citri. Virology 156: 305–312.

Lee YJ, Yi H, Kim WJ et al. (2009) Fabricating genetically engineered high‐power lithium‐ion batteries using multiple virus genes. Science 324: 1051–1055.

Lin NT, Liu TJ, Lee TC et al. (1999) The adsorption protein genes of Xanthomonas campestris filamentous phages determining host specificity. Journal of Bacteriology 181: 2465–2471.

Lowman HB, Bass SH, Simpson N and Wells JA (1991) Selecting high‐affinity binding proteins by monovalent phage display. Biochemistry 30: 10832–10838.

Mao C, Solis D, Reiss B et al. (2004) Virus‐based toolkit for the directed synthesis of magnetic and semiconducting nanowires. Science 303: 213–217.

Marciano DK, Russel M and Simon SM (1999) An aqueous channel for filamentous phage export. Science 284: 1516–1519.

Marvin DA, Welsh LC, Symmons MF, Scott WR and Straus SK (2006) Molecular structure of fd (f1, M13) filamentous bacteriophage refined with respect to X‐ray fibre diffraction and solid‐state NMR data supports specific models of phage assembly at the bacterial membrane. Journal of Molecular Biology 355: 294–309.

McCafferty J, Griffiths AD, Winter G and Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348: 552–554.

McLeod SM, Kimsey HH, Davis BM and Waldor MK (2005) CTXϕ and Vibrio cholerae: exploring a newly recognized type of phage‐host cell relationship. Molecular Microbiology 57: 347–356.

Michel B and Zinder ND (1989) Translational repression in bacteriophage f1: characterization of the gene V protein target on the gene II mRNA. Proceedings of the National Academy of Sciences of the USA 86: 4002–4006.

Model P and Russel M (1988) Filamentous Bacteriophage. In: Calendar R (ed.) The Bacteriophages, pp. 375–456. New York: Plenum Publishing.

Model P, Jovanovic G and Dworkin J (1997) The E. coli phage shock protein operon. Molecular Microbiology 24: 255–261.

Mullen LM, Nair SP, Ward JM, Rycroft AN and Henderson B (2006) Phage display in the study of infectious diseases. Trends Microbiol 14: 141–147.

Park SH, Marassi FM, Black D and Opella SJ (2010) Structure and dynamics of the membrane‐bound form of Pf1 coat protein: implications of structural rearrangement for virus assembly. Biophysics Journal 99: 1465–1474.

Rakonjac J, Feng JN and Model P (1999) Filamentous phage are released from the bacterial membrane by a two‐step mechanism involving a short C‐terminal fragment of pIII. Journal of Molecular Biology 289: 1253–1265.

Rice SA, Tan CH, Mikkelsen PJ et al. (2009) The biofilm life cycle and virulence of Pseudomonas aeruginosa are dependent on a filamentous prophage. ISME J 3: 271–282.

Riechmann L and Holliger P (1997) The C‐terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli. Cell 90: 351–360.

Russel M (1993) Protein‐protein interactions during filamentous phage assembly. Journal of Molecular Biology 231: 689–697.

Russel M and Model P (1989) Genetic analysis of the filamentous bacteriophage packaging signal and of the proteins that interact with it. Journal of Virology 63: 3284–3295.

Samuelson J, Chen M, Jiang F et al. (2000) YidC mediates membrane protein insertion in bacteria. Nature 406: 637–641.

Scott JK and Smith GP (1990) Searching for peptide ligands with an epitope library. Science 249: 386–390.

Val ME, Bouvier M, Campos J et al. (2005) The single‐stranded genome of phage CTX is the form used for integration into the genome of Vibrio cholerae. Molecular Cell 19: 559–566.

Waldor M and Mekalanos JJ (1996) Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272: 1910–1914.

Webb JS, Lau M and Kjelleberg S (2004) Bacteriophage and phenotypic variation in Pseudomonas aeruginosa biofilm development. Journal of Bacteriology 186: 8066–8073.

Wrighton NC, Farrell FX, Chang R et al. (1996) Small peptides as potent mimetics of the protein hormone erythropoietin. Science 273: 458–464.

Further Reading

Antonara S, Chafel RM, LaFrance M and Coburn J (2007) Borrelia burgdorferi adhesins identified using in vivo phage display. Molecular Microbiology 66: 262–276.

Craig L and Li J (2008) Type IV pili: paradoxes in form and function. Current opinion in structural biology 18: 267–277.

Darwin AJ (2005) The phage‐shock‐protein response. Molecular Microbiology 57: 621–628.

Johnson TL, Abendroth J, Hol WG and Sandkvist M (2006) Type II secretion: from structure to function. FEMS Microbiology Letters 255: 175–186.

La Farina M, Izzo V, Duro G et al. (1987) Intragenomic recombination between homologous regions of genes II and IV promotes formation of bacteriophage f1 miniphages. Nucleic acid Research 15: 7190.

Liu DJ and Day LA (1994) Pf1 virus structure: helical coat protein and DNA with paraxial phosphates. Science 265: 671–674.

Marlovits TC and Stebbins CE (2010) Type III secretion systems shape up as they ship out. Current Opinion in Microbiology 13: 47–52.

Rakonjac J, Bennett NJ, Spagnuolo J, Gagic D and Russel M (2011) Filamentous Bacteriophage: Biology, Phage Display and Nanotechnology Applications. Current Issues in Microbiology 13: 51–76.

Russel M and Model P (2006) Filamentous Phage. In: Calendar RC (ed.) The Bacteriophages, 2nd edn, pp. 146–160. New York: Oxford University Press, Inc.

Silverman PM and Clarke MB (2010) New insights into F‐pilus structure, dynamics, and function. Integrative Biology (Cambridge) 2: 25–31.

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Rakonjac, Jasna(Jul 2012) Filamentous Bacteriophages: Biology and Applications. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000777]