Peer review of: Structure and mechanism of DNA delivery of a gene transfer agent (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Review for NCOMM-19-38620-T 
 
 
The manuscript “Structure and mechanism of DNA delivery of a gene transfer agent” by Bardy et al 
reports on the structure of gene transfer agents in Rhodobacter capsulatus (“RcGTAs”). Overall this is 
a fascinating topic in biology and an important one to study as gene transfer agents are linked to 
pathogenicity and horizontal gene transfer among bacteria. Thus, a mechanistic study of the phage-
like particles themselves is warranted and expected to be impactful to a broad audience. 
 
This study relies almost entirely on cryo-EM to decipher the structure and function of these particles. 
Bardy et al also make use of mass spectrometry and evolutionary relationships among related phage 
proteins for analysis of the particles. Largely, the high-resolution structures reported in this paper are 
beautiful (very nice figures as well!) and I have little concern over the technical rigor over the imaging 
and reconstruction. I do however have some reservations about several conclusions drawn from this 
work without follow up experimentation. In addition, the presentation of the manuscript needs serious 
work in order to be suitable for a broad-audience targeted journal. As it is currently written, the 
results are overly dense and more suited to a heavily structure-based journal. 
 
Specific comments are listed below: 
 
Overall the introduction is well written and clearly states several interesting points about RcGTAs. One 
question I had is how and why starvation leads to expression of RcGTAs? It seems counterintuitive 
that this would be the case as it would require a lot of energy from the host to express and assemble 
the RcGTAs and under starvation conditions there might not be adequate resources available. Can this 
be explained more? 
 
It is very interesting that the capsid size of the RcGTAs can fit essentially a single Rhodobacter gene 
given the volume. What is known about which genes are selectively packaged? This is an area where 
deep sequencing of purified particles could reveal a great deal about the biology of the RcGTAs and 
provide more biological relevance of the structures. 
 
In general, throughout the text several lengthy descriptions of domain boundaries are given and 
details of individual protein domains are described ad nauseum (example is lines 91-98). The HK97 
fold and other phage folds are quite well-established. Technical jargon should be eliminated. One 
suggestion is to list the residue numbers and domain names in the figures (such as in the color-coded 
protein schematics) rather than in the text. Reading these detailed descriptions was tedious and dry 
and did not provide much relevance. 
 
A raw micrograph showing the oblate and isometric heads would be useful to include. 
 
EMDB submission of maps and validation of all models should be required. 
 
The comparison of secondary structure elements between the tape measure protein and the tail 
needle protein from P22 are not convincing (lines 265-269). This is an odd comparison given the 
structures of these two types of phage tails. Is there some labeling or other means of identifying this 
density corresponds to this protein? 
 
Page 9 contains an enormous amount of speculation. I am not convinced there is enough evidence to 
make reasonable conclusions about the peptidase. Can the authors expand on how they came to these 
conclusions? Process of elimination? Copy number? Mistakenly identifying density in complex maps is 
easy to do, so this reviewer would need much more proof of where these proteins residue other than 
the structure alone. Labels? Knockouts? 
 
The overly large MOI of particles used in the image of Figure 6 is also problematic and makes these 
data impossible to accurately analyze. What are the statistics of how many phage particles are bound 
to the cell surface and what is the distribution of orientations? How many images were analyzed? 
There is nothing I could see in the M&M that describes this experiment. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The manuscript by Bardy and co-authors “Structure and mechanism of DNA delivery 1 of a gene 
transfer agent” reports a very interesting and important structural study of a phage-like complex 
produced by the alphaproteobacterium Rhodobacter capsulatus. Some bacteria use phage-like 
complexes (gene transfer agents or GTAs) to mediate horizontal gene transfer. The genetic exchange 
using GTA complexes is linked to bacterial adaptation and evolution. 
 
Bardy and co-authors report the first nearly complete structure of the GTA of Rhodobacter capsulatus 
(RcGTA) obtained by cryo-EM. The organization of the RcGTA reminds a tailed double-stranded DNA 
phage. Each complex is packed with a short piece of DNA (~4,500 kb pairs) from the bacterial 
genome. Apparently, pieces of genome are selected randomly, and they do not contain information on 
the GTA structural elements. The authors of this MS describe a number of interesting facts: overall 
organization of particles has a phage-like appearance, these GTAs have oblate heads, their sizes are 
lesser compared to the sizes of smallest phages from the subfamily Picovirinae of Podoviridae. The 
authors have obtained the complete structure of the capsid, in which capsid proteins, portal proteins 
were identified allowing to reveal points of interactions between them. The authors have determined 
the structure of the tail which represent some sort a hybrid formation between tails of the 
Siphoviridae and Podoviridae phages: the particle has a small tail which is longer compared to 
Podoviridae with a few rings forming the tail tube like in Siphoviridae. Like all phages these phage-like 
particles have rather characteristic organization of the tail with the tail tip and fibers. 
The authors have done considerable work in analysis of essential elements of the complex and 
suggested a hypothesis of its function. This analysis will help to reveal processes of the genome 
exchange, propagation, infection, and evolution related to the development of the bacterial resistance 
to medical drugs. 
 
There are some questions that should be addressed by authors to improve the MS. 
 
What is a difference between the head measure and the tape measure proteins (see the first 
paragraph on page 4)? Why do the authors expect to find a measure protein within the capsid? 
Typically, dsDNA phages do not have additional proteins within the capsids. So far in other studies 
related to phages a head measure proteins were not identified. It would be good to have a definition 
of this protein and what can it define? As it has been shown so far sizes of the heads depend on 
scaffolding proteins, components involved during self-assembly, and genome size. This first paragraph 
(page 4) is not consistent with overall information on the phages and results shown in Fig S25 (A and 
B). Mio- and Siphoviridae families have a separate process for the assemblage of tails, where this tape 
measure protein became essential. This paragraph is not consistent with middle paragraph in page 7. 
The conclusion in the paragraph on page 7 (the last sentence) is unjustified.  
Head spikes. It would be good if the authors will explain, what do they name as spikes and what is a 
base protein. Is it located on the outer surface of the capsid? Where are the fibers located? It seems 
that , they are not resolved? 
 
The authors have to explain themselves: “A dodecamer of portal protein subunits (Rcc01684, g3) 
replaces one of the two pentamers of major capsid proteins positioned on the fivefold axis of the 
oblate RcGTA capsid”. The sentence is very confusing: although the capsid of the GTA is oblate, it still 
has 12 5-fold vertices. It is the overall structure that does not have 12 5-fold axis of symmetry, but 
only one. It is the same for all tailed phages. So which one is occupied by the postal complex? 
 
The authors did not provide an explanation, how they have obtained the structure of the portal protein 
complex with imposed 12f symmetry and what they have seen in the structure obtained without any 
symmetry. The indication of interactions between capsids proteins and the portal complex within the 
capsid where a strong mismatch of symmetries takes place remains unconvincing. To justify changes 
in symmetry the authors have to provide both figures with the fittings and assessments of differences 
between different conformations. Possibly, the authors have to find at least three different 
conformations for the capsid protein or even all five can be different. Possibly the contacts have to 
break 12f symmetry of the portal complex as well. 
 
Stopper proteins do not regulate the release of the dsDNA, or the authors have to express their ideas 
a bit clearer. As it has been shown previously by other authors a role of the stopper proteins is to lock 
the last end of the dsDNA packaged into the procapsid prior attachment of the tail to the phage head. 
The release of the genome into the bacteria is regulated by signals that come from the distal tail tip 
towards the heat-to-tail connector. 
 
It would be interesting to know, how the authors have defined local symmetries of the tail 
components. According to publications by other groups (Leiman, Morais, Conway) there are significant 
differences in symmetries of the tail components from 3f to 6f (including 4 and 5f symmetries). How 
the symmetries were assessed for the presented structures? 
 
I am not sure that it can be said “Coordination of a putative cation by side chains of Asp210 of major 
capsid proteins and Glu12 of base proteins strengthens the attachment of head fibers to the RcGTA 
head.” What kind of indications were found for the presence of cations in the structure? It seems that 
the structure of the head fibers was not well defined. The authors did not present any clear evidence 
for that. 
 
Format of figures is a bit confusing: even in one figure the same elements are shown at different 
scales making meaningful comparisons rather difficult. 
 
My impression that the authors have done an important study and found many interesting features of 
the analysed GTA particles. The study is important, but the authors did not achieve the best resolution 
that would allow them to trace all proteins de novo and to make strong conclusions on fine features of 
the complex. It is not an easy job. Unfortunately, the authors did not provide detail information how 
the polypeptide chains were traced and possibly some parts of them were represented by 
polyalanines? 
 
The authors have done excellent structural job but done some overfitting and overinterpretation. The 
logistic of the MS should be improved so the repetitions could be removed, the number of 
supplementary figures should be reduced, and they should be made more concise to help to 
understand what it similar and what is special between phage-like particles and picovirinae phages. It 
would be important to clarify a biological role and impact of such particles designed by Nature. 
 
It is strongly recommended to do proofreading for English of the MS. 
 
 
Minor comments 
 
It seems that the authors submitted the MS to another journal, then they have modified it and 
numbering of figures became confusing: supplementary figures are starting from number 5, and the 
suppl. figures 1-4 are coming much later. That caused some disruptions in logistic: the topic of the 
tape measure protein was raised in different parts of the MS Page 4. 
 
Separation of the portal protein of the certain type of domains has been done not in 2015 for the T4 
portal protein, but at least in 2007 or even earlier in 2003. Please use the correct reference. 
 
Why is it so important to show some distances between amino acids and not to show them as fitted in 
the EM maps? The distance were actually not discussed in the text. 
 
Figure 3D -> difficult to understand where are points of interactions between portal, capsid and 
adaptor proteins; 3E -> the points of interactions are not clear. Was it possible to reveal interactions 
between certain amino acids?