Filamentous Bacteriophages: Biology and Applications


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