Peer review of: “Gene accordions” cause genotypic and phenotypic heterogeneity in clonal populations of (2020)



REVIEWERS' COMMENTS: 

Reviewer #4 (Remarks to the Author): 

It was a pleasure reading the revised manuscript by Belikova et al. As I was asked to review a 
revision of the paper, I focused on whether the authors responded well to reviewers’ comments, 
whether there are any major problems that had been overlooked before, and also whether there 
are some minor errors left. Overall, I find the study of high general importance. It provides 
convincing evidence for the importance of scalable gene amplification or gene accordions during 
host adaptation of Staph. aureus and the likely underlying reasons. The study was very well set-
up, starting with a broad description of the phenomenon in clinical isolates, followed by a detailed 
experimental analysis using evolution experiments, functional genetic analysis, and also infection 
experiments in mice. The specific results are based on well designed experiments and a sound 
statistical analysis of the data. The conclusions drawn are very well justified by the data. The 
functional importance of such gene amplifications is usually unknown and the phenomenon itself 
often neglected in current characterizations of human pathogens. Thus, the study by Belikova et 
al. fills an important knowledge gap and consequently it is highly suited for publication in Nature 
Communications. 

I do not see any major problems in the revised manuscript. Moreover, in my opinion, the authors 
did address the previously raised concerns very well. I only have two minor comments: 

1) Figure 2: The authors should briefly add an explanation on why ciprofloxacin was used in the 
experiments. This is not clear. Moreover, Fig. 2B needs further explanation. It is not clear what the 
curves should highlight and what exactly is shown in the two rows of the Western blot. 
2) Lines 248 onwards: To enhance clarity, the authors should in this part of the manuscript explain 
the rationale of their experiment in more detail. In particular, they should explain why the 
supernatant of the bacterial cultures is used to assess the immunestimulatory effect of the csa1-
locus encoded lipoproteins. Is it known that these lipoproteins are shedded and thus available in 
the supernatant? 

Reviewer #5 (Remarks to the Author): 

The study by Balikova et al. analyses tandem amplifications or deletions of genes in natural 
bacterial populations and bacterial lipoproteins potential to modulate host pathogen interactions 
through amplifications/segregations. 
The authors show that tandem amplifications of the gene csa1 in S. aureus genome sequences are 
common. However, for other genes deletions are dominating. They show that amplifications and 
segregations of the tandem amplifications of csa1 is happening at a high frequency both in vivo 
and in vitro. With the additional negative controls (inactivated csa1 gene, (FS)) the experiments 
are convincingly showing that the altered copy number of csa1 changes the gene expression and 
as such modulates the immune response. 
The manuscript is well written and has been improved with additional crucial experiments as 
suggested by previous reviewers (inactivation of csa1). This paper will add to the field of unstable 
amplifications that transiently changes gene expression. 

Minor points. 
1. The authors state that they found several hotspots of variation, gene copy number (stated in 
the abstract). According to the supplemental information most of the hotspots contained deletions 
of genes and very few amplifications with the exception of csa1. As deletions are evolutionary 
dead ends when it comes to amplification of tandem repeats, which also the authors recognize, the 
impact of multiple hotspots changing gene expression is over stretching the message as written in 

the abstract. 

2. The sentence “Interestingly, we observed several spontaneous segregation events where by 
cells of the culture had undergone deletions that led to a drastic reduction in copy number (eg 
E26A and E26B in Fig. 5A)” make no sense to me. 
I. I can’t find information on E26A and E26B in Fig. 5A, do they mean Fig. 6A. 
II. I don’t see reduction of E26B in Fig 6A (if that is the correct figure), if anything an increase is 
detected. Maybe they mean L38A?