Filamentous phages: masters of a microbial sharing economy

Abstract

Bacteriophage ("bacteria eaters") or phage is the collective term for viruses that infect bacteria. While most phages are pathogens that kill their bacterial hosts, the filamentous phages of the sub-class Inoviridae live in cooperative relationships with their bacterial hosts, akin to the principal behaviours found in the modern-day sharing economy: peer-to-peer support, to offset any burden. Filamentous phages impose very little burden on bacteria and offset this by providing service to help build better biofilms, or provision of toxins and other factors that increase virulence, or modified behaviours that provide novel motile activity to their bacterial hosts. Past, present and future biotechnology applications have been built on this phage-host cooperativity, including DNA sequencing technology, tools for genetic engineering and molecular analysis of gene expression and protein production, and phage-display technologies for screening protein-ligand and protein-protein interactions. With the explosion of genome and metagenome sequencing surveys around the world, we are coming to realize that our knowledge of filamentous phage diversity remains at a tip-of-the-iceberg stage, promising that new biology and biotechnology are soon to come.

Keywords: Zot; filamentous phage; phage; procoat protein; secretin.

Figure 1. Filamentous phages: classification and applications in biotechnology

(A) Bacterial and Archaeal virus sub‐families are represented and grouped based on their Baltimore classification. Relative sizes and symmetries are approximate. (B) Schematic representation of the Escherichia coli Ff phage showing the overall architecture and copy number of the structural proteins. (C) In phage‐display screening, a natural or synthetic DNA library is cloned between the signal peptide and mature pIII‐encoding gene on a phagemid vector containing an Ff origin of replication, a plasmid origin of replication, and a selectable marker. The phagemid pool is transformed into E. coli infected with a helper phage containing a compromised Ff origin. The helper phage produces all the machinery required for phage replication and assembly, and the phagemid produces modified pIII capsids. Phages are assembled and secreted with a subpopulation of the pIII capsid proteins containing the insert. The phage library them undergoes multiple rounds of “panning”: (i) phages are applied to a matrix with immobilized ligand or target, those phages displaying peptides which bind to or are recognized by the target/ligand are bound while the non‐binding phages are washed away, and then, (ii) bound phages are eluted and used to infect E. coli cells, which are then pooled and infected with a helper phage to amplify the library and produce phages for a subsequent round of panning. Multiple cycles of panning can produce peptides with increased affinity.

Figure 2. Diversity of filamentous phage genomes

(A) Schematic representation of filamentous phage genomes: for each gene identified in the genome, the putative function is noted either based on experimental evidence, inferred from sequence homology, or based on conserved domain predictions. Scale bare represents genome size in nucleotide base pairs. (B) Protein sequence similarity network plot of all predicted open‐reading frames from 56 filamentous phage genomes. The great proportion of orphan proteins in this plot demonstrates that at the protein sequence level, there is a very high degree of diversity in filamentous phages. Each circle node represents a sequence, and each connecting line represents a BLAST score better than 1e‐5. Identical proteins are collapsed into one circle with the size representing the number of proteins denoted. Representative species are coloured as shown, and the identity of the Ff proteins is annotated in the plot.

Figure 3. Phylogenetic tree of filamentous phages

Phylogenetic tree built of the conserved pI homologues of known filamentous phages and prophages. Alignments were calculated with mafft generated (L‐INS‐i option), and sites for tree inference chosen using trimal (automated1). The tree was calculated with RAxML “PROTGAMMAAUTO” criteria (final model LG) and “autoMRE” bootstrap convergence test and midpoint rooted 236, 237, 238. Clades are coloured as described in the text, and leaves are coloured based on their ICTV genera classification.

Figure 4. Lifecycle of the archetypical filamentous Ff phage

(A) In the initial stage of phage attachment, the N2 domain of pIII binds to the tip of the F‐pilus on the surface of the bacterial cell. Upon F‐pilus retraction, the pIII/pVI terminus of the phage would be brought into periplasm of the host cell. The N1 domain of pIII binds to the host protein TolA in the TolQRA complex in the inner membrane. The next stage, which has not been characterized, would need to result in phage disassembly and injection into the cytoplasm of the ssDNA genome termed the “infective form” (IF). (B) Phage replication ensues through recruitment of the host RNA polymerase to a hairpin at the negative (−) origin of replication, resulting in synthesis of a short RNA primer. The positive (+) strand is then extended by the host DNA polymerase III, generating a double‐stranded phage genome termed the “replicative form” (RF). Early in the infection, this can serve as a template for host RNA polymerase to generate phage mRNA, to be translated into phage proteins. The phage protein pII binds to the + origin of replication and nicks the + strand, and the resulting 3′ end is extended by host DNA polymerase III displacing the “old”  + strand. Upon one full cycle, pII cleaves and ligates the + strands resulting in a single‐stranded IF and a double‐stranded RF. The RF can then undergo multiple rounds of rolling circle replication to replicate the phage genome and also serve as a template for transcription and translation of phage proteins. Later in the infection, single‐stranded IF is coated by phage protein pV, leaving the packaging signal‐free in preparation for secretion. (inset) A schematic representation of the phage intergenic region containing the packaging signal, the – origin of replication and the + origin of replication is shown. (C) Structural phage proteins and phage proteins required for assembly and secretion are shuttled to the inner membrane and processed by the SecYEG, YidC and signal peptidase machinery. The packaging signal hairpin of the pV‐coated ssDNA is bound by the minor capsid proteins pVII and pIX and recognized by the pI/pXI IM assembly proteins. As the emerging ssDNA traverses the inner membrane, pV is removed and replaced by the membrane‐embedded major capsid protein pVIII. As pVIII is added to the emerging phage, the tip is forced outwards through the oligomeric secretin‐gated channel pIV. The terminal phage capsid proteins pIII and pVI detect and cap the end of the nascent phage allowing its release from the host cell. Host proteins are represented as various shades of purple. Phage proteins involved in DNA replication and packaging are represented by shades of blue. Phage proteins involved in secretion are shown as shades of orange. Structural phage proteins are shown as shades of green. (inset) Phage proteins which interact with the bacterial membranes are shown. Topogenic signal peptides and transmembrane regions are annotated.

Figure 5. Methods of filamentous phage host chromosome integration and excision

Methods for filamentous prophage integration into the host chromosome are shown. Top: Host‐mediated (XerCD) integration via two methods. Vibrio phage VJG uses a reversible integration—a dsDNA RF phage genome with an attP site is recognized by the host XerCD recombinase which mediates homologous recombination at the dif site on the host chromosome. The prophage can be excised by XerCD‐mediated recombination at the resulting attL and attR sites. Vibrio phage CTXφ uses an irreversible integration—XerCD recognizes an attP site formed by a hairpin in the ssDNA phage genome and mediates homologous recombination at the dif site on the host chromosome (and typically a satellite phage). Due to nature of the attP hairpin, the resulting AttL site on the prophage is defective and thus cannot be excised by XerCD. Replication of the resulting prophage is inactivated by a regulatory loop involving the phage‐encoded repressor RtsR (R), the host repressor LexA (L) and the satellite activator RtsC (C). Upon activation by the host SOS response, LexA is degraded and the positive regulator RtsC is produced and binds to the RtsR repressor allowing expression of the phage replication protein RtsA (functionally equivalent of pII). RtsA binds to the + ori on the prophage genome and acts in an analogous way to that of pII on RF circular DNA. The resulting phage ssDNA is amplified and packaged as described in Fig 4B. In examples of phage‐mediated integration (bottom), Pseudomonas phage Pf4 uses a phage‐encoded integrase to reversibly integrate itself into the Gly tRNA site of the host chromosome and Ralstonia phage RSM1 uses a phage‐encoded resolvase to reversibly integrate into Ser tRNA site on the host chromosome, while Neisseria phage MDAφ uses a phage‐mediated transposase to integrate at a 20‐bp repeat region (dRS3) on the host chromosome.

Figure 6. Ff discovery through gene signatures in prokaryote (host) chromosomes

Zot domains are widely distributed throughout prokaryotic organisms including Gram‐negative, Gram‐positive and Archaeal organisms. Proteins from the UniRef90 database (representing 2,205 UniProtKB entries) with predicted “Zot” domains (PF05707) are represented in a phylogenetic tree. The taxonomic kingdom (or bacterial superphylum) is indicated in the outer ring. Branches/clades containing the known filamentous phages are coloured according to clades described in Fig 3. PF05707 domains were aligned and a tree was built with RAxML “PROTGAMMAAUTO” criteria (final model used = BLOSUM62) and “autoMRE_ING” bootstrap convergence test and midpoint rooted.