The MpsAB Bicarbonate Transporter Is Superior to Carbonic Anhydrase in Biofilm-Forming Bacteria with Limited CO2 Diffusion

ABSTRACT CO2 and bicarbonate are required for carboxylation reactions, which are essential in most bacteria. To provide the cells with sufficient CO2, there exist two dissolved inorganic carbon supply (DICS) systems: the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA). Recently, it has been shown that MpsAB is a bicarbonate transporter that is present not only in photo- and autotrophic bacteria, but also in a diverse range of nonautotrophic microorganisms. Since the two systems rarely coexist in a species but are interchangeable, we investigated what advantages the one system might have over the other. Using the genus Staphylococcus as a model, we deleted the CA gene can in Staphylococcus carnosus and mpsABC genes in Staphylococcus aureus. Deletion of the respective gene in one or the other species led to growth inhibition that could only be reversed by CO2 supplementation. While the S. carnosus Δcan mutant could be fully complemented with mpsABC, the S. aureus ΔmpsABC mutant was only partially complemented by can, suggesting that MpsAB outperforms CA. Interestingly, we provide evidence that mucus biofilm formation such as that involving polysaccharide intercellular adhesin (PIA) impedes the diffusion of CO2 into cells, making MpsAB more advantageous in biofilm-producing strains or species. Coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. In conclusion, the distribution of MpsAB or CA in bacteria does not appear to be random as expression of bicarbonate transporters provides an advantage where diffusion of CO2 is impeded. IMPORTANCE CO2 and bicarbonate are required for carboxylation reactions in central metabolism and biosynthesis of small molecules in all bacteria. This is achieved by two different systems for dissolved inorganic carbon supply (DICS): these are the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA), but both rarely coexist in a given species. Here, we compared both systems and demonstrate that the distribution of MpsAB and/or CA within the phylum Firmicutes is apparently not random. The bicarbonate transporter MpsAB has an advantage in species where CO2 diffusion is hampered—for instance, in mucus- and biofilm-forming bacteria. However, coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. Given the clinical relevance of Staphylococcus in the medical environment, such findings contribute to the understanding of bacterial metabolism and thus are crucial for exploration of potential targets for antimicrobials. The knowledge gained here as exemplified by staphylococcal species could be extended to other pathogenic bacteria.

and elsewhere-eukaryotic and prokaryotic CA aren't the best descriptors, despite the PFam names, as there are representatives from each group that have the 'wrong' CA. I'd keep the Pfam numbers (e.g., PFam00484) but ditch the names for ones that more adequately describe the evolutionary history of these enzymes. 'Prokaryotic CA' enzymes are beta-class carbonic anhydrases; 'Eukaryotic CA' enzymes are alpha-class carbonic anhydrases.
Did you look for gamma CA? Line 531 did you mean "The respective colonies harboring the right genes"? Not sure what is meant here Line 588 Replace 'expect' with 'except' Line 598 Replace 'avoid the cells clump formation due to possible biofilm' with 'to disrupt clumps' Line 642 replace 'use' with 'used' Supplemental figure 1 caption-A and B are mislabeled; I think the "A" in the figure is skipped in the caption. Also rephrase "CA is mostly point to the right" as "CA genes mostly point to the right" Line 191 "No significant homology to any other proteins was found, implying that only a single CA is present in each strain." Since the different classes of carbonic anhydrase have independent evolutionary origins, using a BLASTp search with a beta CA would not get any alpha CA hits (or vice versa, or gamma CA hits, et cetera). Rephrase this.
Line 206 'is the ligation of the zinc active site with sulfur atoms..." could be rephrased as "facilitates the ligation of zinc in the active site, with sulfur atoms.. Line 377 why would aerobic organisms have a higher requirement for anaplerotic reactions than anaerobic ones? Is this because the aerobic ones would have a complete oxidative citric acid cycle, while the anaerobic (presumably fermentative ones) would not? If so, clarify for the reader Line 384 there have been a number of good studies on CA in other members of Bacteria; not sure 'few' fits. Other species, just for alpha-CA: Mesorhizobium loti Rhodopseudomonas palustris Helicobacter pylori Thiomicrospira crunogena And others. I think Neisseria might be in there too Plus all the work on carboxysomal carbonic anhydrase There are places in the discussion that could be shortened, that basically repeat results instead of interpreting them. Parts of the paragraph beginning on line 397 could be abbreviated to avoid repeating the results section and refocus the paragraph on interpretation. This is also true for the paragraph beginning on line 446. This could be accomplished by modifying the first few sentences of this paragraph. The same is true for the paragraph beginning on line 465 biofilm-forming bacteria with limited CO2 diffusion" (ID: Spectrum00305-21) was reviewed carefully. The authors paid a detailed attention to both MpsAB and CA in Staphylococcus aureus and S. carnosus to explore their importance in CO2 uptake and bicarbonate transfer. Despite of the great attractions of the subject and performed studies, big questions and doubts have raised as following that must be solved by the authors of this study: 1-Line 80: CO2/bicarbonate (HCO3−)?!!! It must be without CO2. 2-Line 81: the correct is "eukaryotes". 3-Line 89: "CAs are a cytoplasmic enzyme". Generally, CAs can be localized in other subcellular locations like membrane, mitochondria, and nucleus as well as cytoplasm. In addition, CAs are classified to eight evolutionary families, not classes. In addition, it would be better to state what kind of CA families are present in prokaryotes. You can use these Refs for more information and citation: "PMID: 29802189 and PMID: 32393172". 4-Line 116: "Gammaproteobacteria" should be non-italic. 5-Line 116 and line 118: we have two different form of writing: "Gammaproteobacteria" and "γproteobacterial". The text must be uniform. One form the beginning to end. 6-Line 124: "Firmicutes" must be non-italic. 7-Line 128: "S. aureus possess only the MpsAB transporter, but no CA". How did you find there is no CA in S. aureus? This statement was mentioned again in lines 134-135. Again, there is this mistake in Table 3 Even, it was mentioned there is no CA in S. aureus in a "Nature Communication" paper that is not correct: Table 1 in "PMID: 31399577", which the first author and the corresponding author are similar to this manuscript. A big mistake was published in the "Nature Communication" in 2019 and another one has submitted to a journal from ASM now. 8-Line 136: the CA from Staphylococcus carnosus belongs to what CA family that was mentioned in Figure 1 as well? Why? Please indicate in the manuscript.

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Dear Editor,
The manuscript entitle "The MpsAB bicarbonate transporter is superior to carbonic anhydrase in biofilm-forming bacteria with limited CO2 diffusion" (ID: Spectrum00305-21) was reviewed carefully. The authors paid a detailed attention to both MpsAB and CA in Staphylococcus aureus and S. carnosus to explore their importance in CO2 uptake and bicarbonate transfer. Despite of the great attractions of the subject and performed studies, big questions and doubts have raised as following that must be solved by the authors of this study: 1-Line 80: CO 2 /bicarbonate (HCO 3 − )?!!! It must be without CO 2 . 2-Line 81: the correct is "eukaryotes". 3-Line 89: "CAs are a cytoplasmic enzyme". Generally, CAs can be localized in other subcellular locations like membrane, mitochondria, and nucleus as well as cytoplasm. In addition, CAs are classified to eight evolutionary families, not classes. In addition, it would be better to state what kind of CA families are present in prokaryotes. You can use these Refs for more information and citation: "PMID: 29802189 and PMID: 32393172". 4-Line 116: "Gammaproteobacteria" should be non-italic. 5-Line 116 and line 118: we have two different form of writing: "Gammaproteobacteria" and "γ-proteobacterial". The text must be uniform. One form the beginning to end. 6-Line 124: "Firmicutes" must be non-italic. 7-Line 128: "S. aureus possess only the MpsAB transporter, but no CA". How did you find there is no CA in S. aureus? This statement was mentioned again in lines 134-135. Again, there is this mistake in Table 3. Based on my analyses, there is one beta CA in S. aureus as following: Even, it was mentioned there is no CA in S. aureus in a "Nature Communication" paper that is not correct: Table 1 in "PMID: 31399577", which the first author and the corresponding author are similar to this manuscript. A big mistake was published in the "Nature Communication" in 2019 and another one has submitted to a journal from ASM now. 8-Line 136: the CA from Staphylococcus carnosus belongs to what CA family that was mentioned in Figure 1 as well? Why? Please indicate in the manuscript.
In the current form, this study is not recommended for publication at "Microbiology Spectrum" and even transfer to other sister journals from ASM. The general subject for this manuscript is attractive and can be re-submitted after extensive modification on it.
Best wishes

Reviewer #1 (Comments for the Author):
This manuscript is a very interesting study of the interplay of carbonic anhydrase and dissolved inorganic carbon transporter presence in genomes of Firmicutes. The study does a nice job showing how CA or transporter presence can complement loss of one or the other in site directed mutants, and that cells that produce an elaborate extracellular matrix might be particularly beholden to dissolved inorganic carbon transporters to obtain sufficient dissolved inorganic carbon for growth. Specific suggestions on the manuscript follow: Line 41: carbonic anhydrase doesn't really concentrate inorganic carbon, it merely facilitates its interconversion. Change 'carbon concentration systems' to 'systems for dissolved inorganic carbon supply'?
We really thank you for this suggestion. We also think that 'carbon concentrating system' is not really correct. We changed all according to your suggestion: 'systems for 'dissolved inorganic carbon supply' (DICS). Please see line 40 and other changes highlighted in yellow.
Line 57: replace 'as bicarbonate transporter has an advantage' with 'expressing bicarbonate transporters provides an advantage' Thank you for the suggestion. We have amended it accordingly. Please see line 56.
Line 64 and elsewhere (e.g., line 88): see comment above about line 41 We have replaced 'carbon concentration systems' to 'systems for dissolved inorganic carbon supply' throughout the manuscript. Please see the changes highlighted in yellow.
Line 89 some carbonic anhydrase enzymes are found in the periplasm, and some are membrane-associated Thank you for pointing out the mistake. We have corrected the sentence to "CAs are ubiquitous enzymes which can be found in the mitochondria, cytoplasm, periplasm, membrane or cell wall-associated, carboxysome and also chloroplast in plants (3,4)". Please see line 89.
Line 89 "with a mostly a Zn-binding domain" should be reworded as "most of which have a Zn-binding domain" Thank you for the comment. We have corrected the sentence to "Most of these enzymes have a Zn-binding domain that……. Please see line 91.
Line 91 CA activity, since it can also convert bicarb to CO2, does not trap bicarb in the cytoplasm. One of the early experiments to 'prove' that the dissolved inorganic carbon pool in the cytoplasm of cyanobacteria consisted predominantly of bicarbonate and was out of chemical equilibrium consisted of expressing human CA in a cyanobacterium expressing bicarbonate transporters. When this was done, these mutant cells became "CO2 fountains"---vast quantities of cytoplasmic dissolved inorganic carbon were lost as CO2 Thank you for the comment. We removed the 'trapping part' and rephrase the sentence: CA is important for inorganic carbon fixing-enzymes which utilizes either CO2 or HCO3by interconverting these species to ensure sufficient concentration in the cytoplasm. Please see line 93.
Line 97 "In this process, HCO3-is continuously removed from the chemical equilibrium of the external milieu and at the same time it is continuously replenished." Not sure what is meant here?
We apologized for the confusion. We have rephrased the sentence: Such a transporter transports HCO3 − from the external environment over the membrane into the cytoplasm, where the imported HCO3 − is consumed by the carboxylation reactions. The continuous consumption of HCO3 − in the cytoplasm could induce a suction power to keep the transporter running. In the exterior milieu, the transporter is continuously removing HCO3 − from the CO2 / HCO3 − equilibrium resulting in a permanent replenishment of HCO3 − . Please see line 103. Table S2 and elsewhere-eukaryotic and prokaryotic CA aren't the best descriptors, despite the PFam names, as there are representatives from each group that have the 'wrong' CA. I'd keep the Pfam numbers (e.g., PFam00484) but ditch the names for ones that more adequately describe the evolutionary history of these enzymes. 'Prokaryotic CA' enzymes are beta-class carbonic anhydrases; 'Eukaryotic CA' enzymes are alpha-class carbonic anhydrases.
Thank you for the comment. We agreed, too, the eukaryotic and prokaryotic CA are not the best descriptor but we used the Pfam names just because they are named as such. However, we would follow your suggestion to keep the Pfam numbers for the Tables and put 'Prokaryotic CA' as β-CA instead and 'Eukaryotic CA' as α-CA. Please see the changes highlighted in yellow in table 1, Figure S1, table S1 and the manuscript and we included a sentence in line 166 regarding this.
Did you look for gamma CA?
Yes. We blasted the  and -CAs listed below against S. aureus, S. carnosus and S. pseudintermedius and we did not find any homology. Below are the BLASTp results. As we have reached the limit of supplementary materials (max 10), we did not include it there. However, now we include the info in the manuscript, "For S. carnosus and S. pseudintermedius genomes, we did not find any other homology Line 531 did you mean "The respective colonies harboring the right genes"? Not sure what is meant here We apologized for the error. We have corrected it to: "The respective clones harboring the right genes……Please see line 535.
Line 588 Replace 'expect' with 'except' We apologized for the typo. Please see the correction at line 591.
Line 598 Replace 'avoid the cells clump formation due to possible biofilm' with 'to disrupt clumps' Thank you for the suggestion. Please see line 601.
Line 642 replace 'use' with 'used' We apologized for the mistake. Please see line 645.
Supplemental figure 1 caption-A and B are mislabeled; I think the "A" in the figure is skipped in the caption. Also rephrase "CA is mostly point to the right" as "CA genes mostly point to the right" Thank you for highlighting the errors. We have amended it accordingly. Please see line 33 and 43.
Line 191 "No significant homology to any other proteins was found, implying that only a single CA is present in each strain." Since the different classes of carbonic anhydrase have independent evolutionary origins, using a BLASTp search with a beta CA would not get any alpha CA hits (or vice versa, or gamma CA hits, et cetera). Rephrase this.
Thank you for the comment. We have included '-CA' since the CAs from S. carnosus and S. pseudintermedius cloned in E. colican are from -CA class: "No significant homology to any other proteins was found, implying that only a single -CA is present in each strain." Please see line 202. We have also included a sentence that BLASTp showed no homology with -and -CAs from other bacteria. Please refer to the above reply and line 206. We apologized if the figure is not clear. We mentioned it in the figure legend that lower OD in figure 4C was due to clumps/cell agglutinations. The readings were recorded automatically by a microplate reader. We have now included an arrow with the word 'clumps' in Figure 4C. Please refer to Supplementary Figure 4C and line 130 in the figure legend.
Line 377 why would aerobic organisms have a higher requirement for anaplerotic reactions than anaerobic ones? Is this because the aerobic ones would have a complete oxidative citric acid cycle, while the anaerobic (presumably fermentative ones) would not? If so, clarify for the reader Thank you for the intriguing questions. It gave us another perspective to the interpretation, which probably might be more plausible. we deleted this statement from the manuscript and added another interpretation in the discussion: "In principle, it does not really sense if the bacteria have both a HCO3 − transporter and a CA because the transported HCO3 − will be converted by CA to CO2 which can escape the cells by diffusion. Thus, the benefit of a transporter is mitigated by the presence of a CA. This is exactly what we observed experimentally by co-expressing can in S. aureus (pRB473-canSc) which caused a decreased growth compared to the wild type. Indeed, most of the bacterial species have only one or the other system. Nevertheless, there are a few species which have both systems like some endospore-forming bacilli and clostridia ( There are places in the discussion that could be shortened, that basically repeat results instead of interpreting them. Parts of the paragraph beginning on line 397 could be abbreviated to avoid repeating the results section and refocus the paragraph on interpretation. This is also true for the paragraph beginning on line 446. This could be accomplished by modifying the first few sentences of this paragraph. The same is true for the paragraph beginning on line 465 We have shortened the Discussion as suggestion. Please see changes highlighted in yellow from line 417-434, 459-469 and 471-474. Line 459 "attributed to the evolution factor" needs to be rephrased. I would delete "can be attributed to the evolution factor, for example mpsAB can be acquired for group (iv) by horizontal gee transfer" and replace it with "could be attributed to acquisition of mpsAB by horizontal gene transfer" Thank you for the suggestion. We have amended it as suggested: The lack of a strict correlation between the last two groups could be attributed to acquisition of mpsAB or ica by horizontal gene transfer. Please see line 464.

Reviewer #2 (Comments for the Author):
The manuscript entitle "The MpsAB bicarbonate transporter is superior to carbonic anhydrase in biofilm-forming bacteria with limited CO2 diffusion" (ID: Spectrum00305-21) was reviewed carefully. The authors paid a detailed attention to both MpsAB and CA in Staphylococcus aureus and S. carnosus to explore their importance in CO2 uptake and bicarbonate transfer. Despite of the great attractions of the subject and performed studies, big questions and doubts have raised as following that must be solved by the authors of this study: 1-Line 80: CO2/bicarbonate (HCO3−)?!!! It must be without CO2.
We apologized for the confusion. We have corrected the sentence to be "CO2 and bicarbonate (HCO3 − )". Please see line 38, 61 and 80.
We have corrected the typo. Please see line 81.
3-Line 89: "CAs are a cytoplasmic enzyme". Generally, CAs can be localized in other subcellular locations like membrane, mitochondria, and nucleus as well as cytoplasm.
Thank you for pointing out the mistake. We have corrected the sentence to "CAs are ubiquitous enzymes which can be found in the mitochondria, cytoplasm, periplasm, membrane or cell wall-associated, carboxysome and also chloroplast in plants (3,4)". Please see line 89.
In addition, CAs are classified to eight evolutionary families, not classes. In addition, it would be better to state what kind of CA families are present in prokaryotes. You can use these Refs for more information and citation: "PMID: 29802189 and PMID: 32393172".
Thank you for the suggestion. CA classes and families are used interchangeably in most of the publications. In fact, one of the most cited publication on CA, "Prokaryotic carbonic anhydrases" by Smith and Ferry (2000) used the term "class" to describe the different groups of CA. We have added the sentence "To date, CAs from eight evolutionary distinct families have been reported (α, β, γ, δ, ζ, η, θ, and ι) (8-11)". Please see line 95-98. 4-Line 116: "Gammaproteobacteria" should be non-italic.
We have corrected the mistake. Please see line 123.
5-Line 116 and line 118: we have two different form of writing: "Gammaproteobacteria" and "γ-proteobacterial". The text must be uniform. One form the beginning to end.
Thank you for the comment. We have standardized the term "gamma" as γ in the text. Please see line 123 and 125. 6-Line 124: "Firmicutes" must be non-italic.
Thank you for the comment. We have amended it accordingly throughout the manuscript. Please see the changes highlighted in yellow. 7-Line 128: "S. aureus possess only the MpsAB transporter, but no CA". How did you find there is no CA in S. aureus? This statement was mentioned again in lines 134-135. Again, there is this mistake in Table 3.
As mentioned in the manuscript (line 367-381), we concluded that there is no CA in S. aureus based on our extensive search using 209 finished genomes sequences in IMG/G database. Since we confirmed the presence of an functional CA in S. carnosus in this work, we blasted this sequence against all 209 genomes sequences but did not find any similarity. We also searched this database for any protein annotated as CA or putative CA and also searched on the basis of two Pfam motifs related to CA but no similarity was found.
In addition, we also blasted the S. carnosus CA against S. aureus in NCBI database and found 2 hits, Sequence ID: SPZ78436.1 (193 amino acids) and Sequence ID: SPZ78435.1 (61 amino acids) annotated as CA from S. aureus. SPZ78436.1 is from a S. aureus strain NCTC12981 which is not fully assembled yet. A BLAST of this protein on NCBI only showed ONE 100% hit with its own sequence (S. aureus) while there are multiple hits from other Staphylococcus species. The highest similarity is from S. schleiferi which covers 69% of the length with 100% identity (e value 5e-94). The second protein, SPZ78435.1 also originates from the same strain, NCTC12981. A BLASTp of this protein also revealed that it has ONE 100% hit with its own sequence (S. aureus) while the multiple hits from other Staphylococcus species, the highest similarity from S. coagulans (86% coverage with 98% identity, e value 1e-27). Clearly, this shows that the protein sequences from unassembled sequences are not accurate and unreliable and therefore we ignored it. For this reason, we restricted our bioinformatics analyses to only fully assembled genomes. We did not simply take a protein sequence from NCBI database but from finished genomes and validated reference genome like S. aureus NCTC 8325 instead. We also included multiple sequence alignment with experimentally proven CAs (Figure 1), gene synteny comparison ( Figure S1) and also phylogenetic analysis ( Figure S7) to support our findings. Furthermore, we have also blasted the genome of S. aureus against the  and -CAs from bacteria in which the CAs activity was experimentally proven. Please refer to the reply for reviewer #1 for the BLASTp results. To prove our point mentioned above, we used the 4 CAs provided by reviewer #2 and performed an extensive BLASTp. First, we blasted all 4 protein sequences from all 259 finished genomes of S. aureus in the IMG/G database which was updated recently. We used this database instead of NCBI because it is more organized to perform search and we could select the exact strains from either finished genomes, permanent draft or drafts. This enabled us to get more 'hits' compared to NCBI which is limited the first 100 sequences producing significant alignments. All 259 genomes showed no identity at all (no hits) for either MVW54107.1, NGG14433.1 and NGB42162.1. The finding that there is no similarity in any of the strains already raised doubts about the authenticity of the protein sequences.
With WP_094666538.1, all 259 strains showed low identity, which was 33% with 40/122 amino acids aligned. These proteins were annotated as either acetyltransferase (isoleucine patch superfamily), acetyltransferase-like (isoleucine patch superfamily), galactoside O-acetyltransferase or hypothetical protein because they have the related COG, KOG or Pfam motifs. If WP_094666538.1 is indeed from S. aureus or even if it is wrongly annotated, it would not be found in such a low identity in all 259 finished genomes, including some reference genomes. For example, one of the protein of our bicarbonate transporter operon, MpsB which is annotated as uncharacterized protein or YbcC family protein (WP_000211540. This strain is wrongly annotated in NCBI database, which gave us "false positive" hits in our BLAST because S. sciuri, but not S. aureus has a CA according our analysis (Table 1 &  Table 3).
Given the fact that not a single strain out of 4819 strains has a reasonable protein identity with any of the CA protein sequences provided by reviewer #2, we proceeded to examine the authenticity of these sequences. Even, it was mentioned there is no CA in S. aureus in a "Nature Communication" paper that is not correct: Table 1 in "PMID: 31399577", which the first author and the corresponding author are similar to this manuscript. A big mistake was published in the "Nature Communication" in 2019 and another one has submitted to a journal from ASM now.
We did not deny that the publication PMID: 31399577 is not from our group. Our current manuscript clearly mentioned that this is a follow-up study of our previous work in PMID: 31399577 (line 137) and therefore the same authors appeared in the manuscript. Our earlier paper PMID: 31399577 stated that the presence of CAs are inferred based on the occurrence of Pfam motif related to CA. In the present manuscript, we supplemented the presence of CA based on Pfam motif with similarity with CA from S. carnosus. As an example, we took the Staphylococcus strains listed in Table S1 and confirmed it with BLASTp of CA from S. carnosus (WP_015900702.1). As can be seen from the right column, the presence of the CAs based on Pfam motif correlate with the high percentage of identity with protein sequence of CA from S. carnosus.  :1, 1088:1, -1092:1, , DOI: 10.1080:1, /14756366.2021:1, .1931863 (2021.

Species
This very recent paper by the same authors (no. 3) who followed up the previous study by reporting its inhibition profile with anions and other small molecules known to inhibit CAs. The same recombinant protein as no. 3 was described, which means that they were still using the CA from C0673, which is actually S. sciuri.
All these papers are published in the same journal (J Enzyme Inhib Med Chem).
They are flawed and misleading and should be retracted.
8-Line 136: the CA from Staphylococcus carnosus belongs to what CA family that was mentioned in Figure 1 as well? Why? Please indicate in the manuscript.
We do not understand your comment here. "Line 135-137 merely state: On the other hand, a gene annotated as encoding a putative CA from Staphylococcus carnosus TM300 has yet to be investigated for its physiological function or enzymatic activity." We did, however, mentioned the CA family in line 212: "According to NCBI Conserved Domain database, the S. carnosus CA belongs to the -class and D clade (cd03379)." Figure 1 is a multiple protein sequence alignment using the 2 strains which we used to in our experiment to generate CA mutants, S. carnosus and S. pseudintermedius along with CAs from S. sciuri, Bacillus subtilis, Streptococcus pneumoniae in which we showed the gene synteny in Supplementary Figure 1. We also included E. coli in the alignment because we used the E. coli CA mutant for our complementation experiments, meaning the CA from E. coli and S. carnosus is interchangeable.
In conclusion, among the almost 5000 S. aureus genomes that we searched for all the CA classes, there was in no case a convincing indication that there are such CAs present in S. aureus species. The statement in our "Nature Communication" paper is therefore correct. Furthermore, we have deleted mpsAB in 4 different wellcharacterized S. aureus strains. None of the mpsAB mutants could grow under ambient air, indicating that there is no functional CA. However, all publications in which a CA in S. aureus has been described and analyzed are dubious. We do not want to burden our current manuscript unnecessarily with the correction of these publications. We would address these flaws and mistakes in a short communication/letter to inform the scientific community about these inaccuracies in the journal and also the NCBI database. After careful review of your responses the reviewers comments, your manuscript is now ready for acceptance. I have also consulted with members of our editorial board about your request to submit an accompanying short communication. We agree that there is merit in submitting a manuscript that corrects the literature regarding CA genes in S. aureus. However, we would consider this as a separate manuscript submission, which would be subject to peer review.
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