A packaging signal-binding protein regulates the assembly checkpoint of integrative filamentous phages
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Curated by eLife
eLife assessment
This valuable work describes a new protein factor that is required for filamentous phage assembly. Convincing evidence is provided for the binding of PSB15 to the packaging signal of the single-stranded DNA, Trx, and cardiolipin, and a mechanism for how the phage DNA is targeted to the assembly site in the bacterial inner membrane is presented. The work will be of interest to microbiologists.
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Abstract
Many integrative filamentous phages not only lack Ff coliphage homologues essential for assembly but also have distinct packaging signals (PS). Their encapsidation remains completely uncharacterized to date. Here we report the first evidence of a PS-dependent checkpoint for integrative filamentous phage assembly. Suppressor screening of PS-deficient phages identified an unknown protein, PSB15 ( PS - b inding 15 kDa), crucial for encapsidation. The WAGFXF motif of the PSB15 N-terminus directly binds to PS DNA with conformational change, while suppressor mutations relieve DNA binding specificity constraints to rescue assembly arrest. PSB15 interacts with phospholipid cardiolipin via its basic helix and C-terminus, and recruits PS DNA to the inner membrane (IM). The PSB15-PS complex is released from the IM by interaction between its hydrophobic linker and thioredoxin (Trx), a host protein that is required for Ff assembly but whose mechanisms are still unclear. Live cell imaging shows that thioredoxin and DNA binding regulate the dwelling time of PSB15 at cell poles, suggesting that they both facilitate the dissociation of PSB15 from the IM. Loss of PSB15 or its PS-binding and IM-targeting/dissociation activity compromised virus egress, indicating that the PS/PSB15/Trx complex establishes a regulatory phage assembly checkpoint critical for integrative phage infection and life cycles.
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eLife assessment
This valuable work describes a new protein factor that is required for filamentous phage assembly. Convincing evidence is provided for the binding of PSB15 to the packaging signal of the single-stranded DNA, Trx, and cardiolipin, and a mechanism for how the phage DNA is targeted to the assembly site in the bacterial inner membrane is presented. The work will be of interest to microbiologists.
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Reviewer #1 (Public Review):
Summary:
This work describes a new protein factor required for filamentous phage assembly. The protein PSB15 binds to the packaging signal of the ssDNA, Trx and cardiolipin. A mechanism how the phage DNA is targeted to the assembly site in the bacterial inner membrane is discussed.
Strengths:
The work describes a clever way to detect factors required for phage propagation by looking at the plaque size of pseudorevertants that arise after infection of a phage with a directed mutation in the packaging signal. This led to the detection of a phage protein expressed from ORF9, the PSB15.
The authors convincingly show that PSB15 is expressed in infected cells and can complement a phage with a mutated orf9.
Weaknesses:
Given the fact that the phage LF-UK is not well explored, many open questions should be mentioned …
Reviewer #1 (Public Review):
Summary:
This work describes a new protein factor required for filamentous phage assembly. The protein PSB15 binds to the packaging signal of the ssDNA, Trx and cardiolipin. A mechanism how the phage DNA is targeted to the assembly site in the bacterial inner membrane is discussed.
Strengths:
The work describes a clever way to detect factors required for phage propagation by looking at the plaque size of pseudorevertants that arise after infection of a phage with a directed mutation in the packaging signal. This led to the detection of a phage protein expressed from ORF9, the PSB15.
The authors convincingly show that PSB15 is expressed in infected cells and can complement a phage with a mutated orf9.
Weaknesses:
Given the fact that the phage LF-UK is not well explored, many open questions should be mentioned in the introduction. For the study, it is important to know if the phageLF-UK has a mimick or homolog of gV and gXI, and if not, whether PSB15 could take their role.
I am not convinced of the proposition of their term "checkpoint". The truth is that the authors do not know the real purpose of PSB15. I do not see an advantage for a checkpoint that only adds an additional step to enter the phage assembly site. There must be a biochemical reason for the action of PSB15. Looking at Figure 7, the step from "Release" to "Loading" is just adding many unknowns, e.g. how to transfer the DNA, how to dispose of PSB15 and Trx? Also, in the previous step are three question marks that do not add any solid information.
The in vivo study of subcellular localization is very questionable. Why is there a single fluorescent dot if there are thousands of PSB15 molecules expressed in the cell? I have my doubts that the conclusions the authors make here are correct and meaningful. The movies do not add anything significant.
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Reviewer #2 (Public Review):
Secretion of the prototypical F-associated filamentous phage (Ff) of E. coli depends on the selective binding of a hairpin (the packaging signal, PS) by two phage encoded protein, pVII and pIX. PVII and pIX target the PS to IM channels formed by pI and pIV. However, integrative filamentous phages lack a homologue of pIX and pIV, and many of them also lack a homologue of pVII, raising questions on the assembly and secretion of new phages. In the manuscript, Yueh et al. present the identification of a phage-encoded protein, PSB15, which binds to the PS signal of a Xanthomonas integrative filamentous phage, ΦLf-UK. They showed that PSB15 is required for viral assembly and is conserved in several other integrative filamentous phages. They further analyzed how PSB15 binds to PS and demonstrated that it associates …
Reviewer #2 (Public Review):
Secretion of the prototypical F-associated filamentous phage (Ff) of E. coli depends on the selective binding of a hairpin (the packaging signal, PS) by two phage encoded protein, pVII and pIX. PVII and pIX target the PS to IM channels formed by pI and pIV. However, integrative filamentous phages lack a homologue of pIX and pIV, and many of them also lack a homologue of pVII, raising questions on the assembly and secretion of new phages. In the manuscript, Yueh et al. present the identification of a phage-encoded protein, PSB15, which binds to the PS signal of a Xanthomonas integrative filamentous phage, ΦLf-UK. They showed that PSB15 is required for viral assembly and is conserved in several other integrative filamentous phages. They further analyzed how PSB15 binds to PS and demonstrated that it associates to the IM, which targets phage DNA to it. Finally, they show that thioredoxin, the only host protein that was found to be essential for Ff secretion, interacts with PSB15 and releases the PSB15-PS complex from the IM. These results are important because they elucidate a major step in the secretion of integrative filamentous phage, and the role of thioredoxin on filamentous phage secretion in general.
I found the data and interpretation convincing. However, the presentation and description are confusing in places because the reader has to juggle between figures. A scheme depicting what is known and unknown in the integration of Ff phages and interactive filamentous phages in the introduction would be useful to the general reader.
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