Filamentous Phage Introduction


Classification of Phages

Phages can be classified in various ways according to the type of genetic material. They can be divided into single-stranded DNA phages, double-stranded DNA phages, single-stranded RNA phages, and double-stranded RNA phages. According to morphology, it can be divided into tail bacteriophage, filamentous bacteriophage, globular bacteriophage, et al.. The filamentous bacteriophage, shown in Figure 1, looks like spaghetti.



Fig 1 Classification of filamentous phage

The Life Cycle of a Filamentous Phage

Although filamentous phages do not rapidly lyse host bacteria and release progeny phages in a short period, as most phages do, they also produce progeny phages and release them into the environment through a specific life cycle. The life cycle of filamentous bacteriophages is different from that of virulent bacteriophages, which are described in three stages: infection, genome replication, and escape from host cells.



Fig 2 Lifecycle of filamentous phage


Almost all filamentous phages can infect a Gram-negative host, so they must cross the host's two membrane barriers. Although the receptor on the cell surface is unknown for most filamentous bacteriophages, some studies have shown that certain bacteriophages can act as receptors for filamentous bacteriophages. Regulated by the pIII protein, the filamentous mycelium can attach to the host cell's pili(Figure 2A). It is hypothesized that the binding of the filamentous milk body to the pili induces the host cell to retract the pili, bringing the filamentous phage to the surface of the host cell. When the pili retract, pIII ends containing viral particles are brought into the periplasmic space to bind to secondary receptors in the host periplasm. Related studies have shown that the secondary receptor is the endometrial misalignment protein TOlA, which extends into the periplasm (Figure 2A). Tola is a component of the TolQRA complex, which controls membrane integrity and invagination during cell division.


Genome Replication


As shown in Figure 2B, the filamentous phage genome is injected into the cytoplasm of the host cell in the form of single-stranded circular DNA(infection form) (IF). The intergenic sequence (IG) located between the gIV and gII genes largely controls the replication of the phage genome. Genome replication of filamentous milk is entirely dependent on the replication mechanism of core bacterial DNA. After the filamentous phage enters the host cell, the host RNA polymerase o70 holoenzyme binds to the negative strand origin that mimics the bacterial -35 and -10 promoter sequences with a much higher affinity than typical bacterial promoters. RNA polymerase begins synthesizing RNA on a single-stranded DNA template but stalls and backtracks in a certain part of the genome and eventually leaves behind a short RNA primer. Host DNA polymerase III holoenzymes extend this primer to produce a negative strand of the genome and a double-stranded copy form of the phage genome (RF). Other regions of the genome can also synthesize RF, but much less efficiently. The host rotates and further processes the RF to form superhelix RF. RF has three key initial functions: First, it is the initial mRNA template that encodes phage proteins, including pII and pX, that are needed to expand the phage genome. Second, it is a template for RF replication. Third, it is a template for IF to copy. The pII protein is a chain transferase that binds to the newly synthesized superhelix RF at the beginning of the replicated positive chain, cutting the positive chain and attaching it to the 5 'end. The free 3 'end can be used as a primer for the host DNA polymerase to synthesize a new positive strand and replace the original positive strand as it evolves.

The Phage is Detached from the Host

In contrast to tailed phages, the physical size of filamentous phages may prevent their assembly inside the cell. Filamentous phages assemble on the cell membrane of the bacteria, while the bacteria actively secret mature phages and do not lyse the bacteria. The newly replicated single-stranded DNAIF genome is coated with DNA-binding protein pV in the cytoplasm, which helps stabilize and expose the "packaging signal" targeting the DNA-protein complex (Figures 2B and C) to the inner membrane of the host cell (Figure 2C). As (initially) mis-signaling proteins, both pI and pXI require bacterial Sec mechanisms to be inserted into the membrane. It is predicted that the cytoplasmic region of DI will act as ATP, powering the assembly and transport of milk. In the absence of other phage proteins or DNA, pI and pXI form complexes with the outer membrane protein pIV. Like other secretins, PIV remains in the outer membrane. While some phages are able to encode their own secretin (pIV), most do not have this ability (Figure 3). Nevertheless, filamentous phages share host cell secretions for their own propagation from bacterial cells, so leaving the host cell by secreting proteins seems to be a common feature of filamentous phages.



Fig 3 Diversity of filamentous phage genomes


Filamentous Bacteriophages are Common


Of the various filamentous phage lineages, only the pI protein is highly conserved and fully recognizable. The pI protein has a conserved Zot domain at its N-terminal (Pfam PFO5707 Zot domain) (Figure 3). The domain is named after the homolog of the pI protein. This congener is known as Zonula Occludens Toxin in Vibrio CTX and is critical for the assembly and release of Vibrio CTX. For filamentous phages and prophages, the Pn-terminal Zot domain (Pfam PFO5707) is unique. Therefore the sequence can be used to evaluate filamentous phages not documented in genomic and metagenomic sequence data.

There are about 2,300 proteins with the PFO5707 domain in the UniProtKB (2018 09) database, almost all of which (>99%) are associated with the genome of prokaryotes and may be derived from filamentous milk. This phenomenon is widespread in a variety of prokaryotes, including various phyla of gram-negative and gram-positive bacteria, as well as archaea. Thus, there may be thousands of unidentified filamentous prophages in the prokaryotic genome library (Figure 4).



Fig 4 There may be thousands of unidentified filamentous prophages in the prokaryotic genome library