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

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The study by Belikova et al. investigates the biological relevance of scalable gene amplification, 
which is a relevant part of the evolutionary ecology of bacteria. Starting with bioinformatics the 
authors identify several putative genomic regions that might be under selection for gene 
amplification in published Staphylococcus aureus genomes. They then examine the biological 
relevance of gene amplifications that span csa1. The authors show that amplifications of this 
region readily occur in vitro and in vivo and can be scaled by the antibiotics 
tetracycline/ciprofloxacin. As bacterial lipoproteins (e.g. Csa) are key players in pathogen-host-
interaction (recognized by TLR2), the authors also suggest that csa copy number variants differ in 
their immuno-stimulatory capacity and that this might be a mechanism to modulate the host 
response. 
While the results are interesting, the study is not as novel as claimed by the authors and several of 
the experiments lack essential negative controls. 

Major Points: 
1. The findings of the study are not quite as novel as stated by the authors (L. 55-57). There are 
numerous studies which show that antibiotics can select for increased copy number (see e.g. ref. 5 
in manuscript). A more recent example comes from Laehnemann et al 2014 who showed that 
doxycline and erythromycin antibiotics stimulates a scalable increase in the copy number of the 
genomic region that spans the multidrug-efflux pump acrAB-tolC. There is also a nice example 
(Elde et al Cell 2012) of how Poxviruses “use” gene amplification to counteract host defences. 

2. The experiments reported show correlations between csa copy number and immune response 
(Fig. 4), and an altered copy number distribution of csa in mice (Fig. 5) that is supposedly 
reflecting adaptation. These experiments lack essential negative controls to separate effects of 
altered Csa levels from (i) indirect effects caused by gene amplification/recombination of the csa 
region or (ii) stochastic effects: 
a. For all experiments a control is needed where the csa gene is inactivated (e.g. by a small in-
frame deletion). 
b. Likewise, a control is needed for the exp. in Fig. 4 to achieve altered Csa levels in another way 
then by gene amplification, i.e. by expression from an inducible promoter. 
c. Finally, for the animal experiments (Fig. 5) a control is needed to determine the copy number 
variation of regions that are not under selection. This control is also required to separate technical 
from biological variation for the analyses in Fig. 1C and Fig. 2. 

3. Regarding the animal experiments in Fig. 5, could not this result simply be the result of a 
stochastic founder effect? Note that mice 5-6 have lower average copy number than input and 
mice 2-3 are not that different from input. To me this looks a bit like the outcome of a Luria-
Delbruck experiment. 

Minor Points: 
1. Fig 1C. The PCR fragment for USFL isolate 165 is not convincing. As this isolate happens to be 
the one with the highest copy number according to qPCR, an experimental validation of these 
results would be helpful. 

2. The lines in Fig. 2C are not labelled, thus the strength of selection in the evolutionary 
experiments cannot be assessed. 

Reviewer #2 (Remarks to the Author): 

Belikova at al. investigated the gene copy number variations in natural populations of bacteria 
which causes genotypic and phenotypic heterogeneity in clonal populations of S. aureus USA300. 
The study shows the expansion and also the contraction of gene arrays occur readily in vitro and in 
vivo with a high various alteration up to more than 100 gene copies in vitro and 30 copies in vivo. 
This is an interesting topic which explains the tremendous ability of prokaryotes to adapt to 
changing environments. In most cases selection of point mutations contribute to adaptation; for 
example respiration defective mutants that lead to small colony variants, SCVs have an advantage 
by causing persistent infections. Here it was shown that also gene duplication or even 
multiplication occurred. With the investigation of some representative host colonization and 
virulence factors such as surface anchored molecule SdrD, the Spl serine proteases and lipoprotein 
gene sca1, the authors pinpoint the gene amplification occurrence during colonization and infection 
which is controlled by the balance of the bacterial benefits and the disadvantage to the host. 
However, the authors should consider the following points: 
Major points: 
1. As far as can be seen there was only one pathogen investigated, S. aureus, and even not 
various S. aureus strains, but only the S. aureus USA300 lineage. This should be then also stated 
in the title. 
2. Lpl3C (SAUSA300_2424) and Lpl2 (SAUSA300_0205) are not lipoprotein genes as they don’t 
contain the right signal peptide with lipobox which is necessary for lgt and lsp function 
(Shahmirzadi 2016 https://www.frontiersin.org/articles/10.3389/fmicb.2016.01404/full). The 
DOLOP is not the up to date program to search for the bacterial lipoproteins (Line 110-111). The 
author should correct them. 
3. csa1A-D, lpl1-9 and tandem lpp (SAUZSA300_2429, SAUSA300_2430) share the highly 
conserved core region 38 aa (Shahmirzadi 2016). However, the author mentioned in Line 394-396 
that primers against csa1 were directed against a highly conserved stretch of the coding sequence 
to allow amplification of a fragment from all four genes (csa1A/B/C/D). The authors should make it 
clear how they can detect the specific genes for csa cluster but other lipoproteins, such as lpl. 
4. USA300 has 3 different tandem lipoprotein clusters: csa cluster (Csa1A/B/C/D), lpl in νsaα 
island, the rest SAUSA300_2429-2430 should be named differently (such as tandem lpp cluster) to 
distinguish them. 
Minor points: 
1. Line 202-203: instead of REF 20, the author should rather mention the Ref 21 and REF (Stoll 
2005) https://www.ncbi.nlm.nih.gov/pubmed/15784587 . 
2. Line 230: ..high csa1 high copy…. 
3. Line 299: instead of REF 21, the author would rather mention the Ref (Nguyen et al. 2017, 
https://www.nature.com/articles/s41467-017-02234-4). 
4. Line 406: there is a typo 
5. Fig1A III part: there should be 4 genes (csa1A to csa1D) but 5 genes are shown. 
6. Fig 2C lacking the explanation of the CIP concentration for each the growth curve. 
7. Fig 2E: lacking the title for Y-axis. 

Reviewer #3 (Remarks to the Author): 

The title refers to gene copy number variation which is usually a term for multiple numbers of 
copies of the same gene in a cell, for example those on plasmids or parts of the genome that have 
replicated to multiple copies. In the text, an alternative is also discussed – gene duplication and 
amplification (GDA), where a second copy of a gene is introduced into the chromosome 
downstream of the original copy. These are two different concepts and the terminology used in the 
title and throughout the manuscript was therefore confusing and conflicting to this reviewer. Are 
we discussing copies or duplicates? What does the term amplification refer to here? 

The first results section identifying gene copy number in staphylococcal chromosomes is not novel. 
The concept here described as GDA is well known in S. aureus chromosomes for a range of genes, 
particularly in the pathogenicity islands (examples Jarraud et al., J immunol 2001; Al Shangiti et 
al., Infect Immun 2004). Although these genes were likely generated by duplication, each 
individual gene has a unique function, eg. Ssls, This would argue that many GDAs are stable, and 
functional. Another similar and well described duplication is in the repetitive binding units within 
genes, such as spaA used for S. aureus typing where one or two duplications arising in a short 
period are seen (Boye & Westh, FEMS Micro Lett 2011). In all cases, the maximum number of 
duplicates is around 20 and does not normally arise from bacterial subculture. This context would 
be useful for the paper. The authors should also consider discussion of the limitation of using short 
read sequencing to estimate gene copy number. 

The novel Results begin by focusing on a single gene, csa1 and showing that GDA duplicates can 
be seen in different isolates. Figure 1 justifies the use of qPCR as a method for identifying the 
numbers of duplicate csa1 genes in isolates with 1-4 duplicates. 

Figure 2a. shows evidence of csa1 duplication after standard growth in the laboratory. qPCR 
amplification suggests over 100 duplications. But I do not see the evidence that csa1 gene has 
duplicated 100 times. SMRT or minIon sequencing showing the duplications would be useful. 
Without this evidence, it suggests that qPCR is not be a robust method for detecting large 
numbers of duplications. 

Since the authors propose multiple genes are duplicated, and this occurs in normal growth 
conditions, why are whole genome sequencing using SMRT technology not identifying isolates with 
100 or more duplications of multiple genes? Could the qPCR method be amplifying mRNA 
generating artificially high counts in the experiments? Or that some regions of the chromosomal 
DNA are replicating at higher frequency? 

In Figure 2b the y-axis shows very low numbers but it is not clear from the legend or the methods 
how this experiment was performed. What is being measured? 

From here on there is such a doubt raised about the validity of the qPCR to detect gene duplication 
that it is not clear how the rest of the data should be interpreted. 

Therefore, while the concept of this paper is interesting, the results are preliminary without the 
necessary robust evidence that the genes described are in fact duplicating.