Anianabacter salinae gen. nov., sp. nov. ASV31^T, a Facultative Alkaliphilic and Extremely Halotolerant Bacterium Isolated from Brine of a Millennial Continental Saltern

Round 1

Reviewer 1 Report

In the manuscript prepared by Maia et al., some bioinformatics analyses and polyphasic analyses of strain ASV31 are described. Please find my comments below.

1. L19. In your opinion and that of extremophiles researchers, how do you define the term "extremely halotolerant"? Is it the maximum it can tolerate or the optimum growth rate?

2. There is a strong emphasis on extreme halotolerance in the title, abstract, introduction, etc. It should be noted, however, that the author did not explain this from a genomic perspective.

3. L95-101. Readers were not informed of the strain in pure culture.

4. L106-109. The set of primers should result in a longer 16S sequence; however, why is it so short? What is the reason for not using sequences from the genome? There may be some slight differences between a complete length and an almost complete length.

5. L141-146. Make sure all chemical names are correctly spelled.

6. How salinity is the sampling site?

7. There are certain types of writing that are not appropriate, such as L213, L379, etc.

8. Section 3.2. Were reference strains analyzed simultaneously with your ASV31? Based on my experience with polyphasic, it is not acceptable to list the characteristics of other stains based on literature data.

9. L260-261. It is unclear to me.

10. Ll403-407. In my opinion, this assumption is risky and not scientifically sound. It is possible to isolate a bacterium with such genes from a completely clean and uncontaminated environment.

11. Section 3.4. What is the significance of this? I suggest that you delete the entire section and provide a proper clue that refers to osmosis stress for which you claimed it is halotolerant.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript is devoted to determining the systematic position of strain ASV31T, which was isolated from solar salterns filled with Santa Engracia spring water. Attribution of the strain to a new species within the family Rhodobacteraceae is beyond doubt. From the authors' data, it is obvious that the new strain is closely related to the Roseobacter group within the family Rhodobacteraceae. The authors try to use the provisions from the article by Wirth and Whitman [40] to determine the systematic position of their new strain and write: an 80% of AAI boundary is needed in the Rhodobacteraceae family for genus determination. However, the above information is misreported, and the meaning of the content of the cited article [40] is strongly distorted by the authors to the opposite. The article [40] cites the findings of Luo et al. [30] that it is difficult to define in discrete boundaries prokaryotic taxa and they exhibit a continuum of AAI values, but not a point boundary. Luo et al. [30] showed that AAI values between members of related but different genera typically vary between 60-80%. That is why the following conclusion (lines 369-371) seems does not agree with bounaries elaborated by Wirth and Whitman [40] and the results presented in the manuscript are not enough to suggest that strain ASV31T may be a new genus within the family Rhodobacteraceae. In fact, the article [40] states that the 16S rRNA gene sequences in the Rhodobacteraceae family lack the resolution for proper phylogenetic reconstruction because the 16S rRNA gene sequence was a poor predictor of phylogeny within this group. That is why Wirth and Whitman [40] suggested and used to identify genome sequences of organisms closely related to members of the original roseobacters, three proteins, RpoC, an uncharacterized acyl-CoA synthetase and SucA because these proteins also had high correlations of 0.94, 0.91 and 0.90, respectively, with the cophenetic distances of the curated core-gene tree. All of the above means that it would be desirable to apply these three proteins to more clearly determine the systematic position of strain ASV31T, especially since ANI 73.9% for ASV31T is sufficient to infer a new species, but AAI 63.5% is not sufficient to infer a new genus and this figure is in the zone of uncertainty.

Despite several references to supplementary data as follows (for example, lines 361, 373-374, 379, 383), I can not find Supplementary material attached to the manuscript on the journal Diversity cite. The article is written in language that is sufficient for understanding the content. Only minor language corrections are needed. Below is a list of suggested corrections where designation ]...[ means deletions and <...> inclusions.

Abstract

30: The suggestion to assign strain ASV31T to a new genus is not valid.

Introduction

60: Is the insersion correct?        "... members of the <Rhodobacteraceae> family"

Methods

129: ]ten[  <10> mL

165-167: ]The Materials and Methods should be described with sufficient details to allow others to replicate and build on the published results.[

Results

230: Enscription in Figure 1 should be corrected: ]Rhoseobacter[  <Roseobacter>

Conclusions

534:  ]stabilised[    <stabilized>

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The authors report on the deep characterization of the strain ASV31T isolated from solar saltern. Analyses included phenotypic and genotypic (including genome analysis) assays to describe ASV31T and highlight its biotechnological potential. The name Anianabacter salinae gen. nov., sp. nov. is proposed. 

Unfortunately, I was not able to find the supplementary materials. In my opinion, the manuscript merites to be published in Diversity, but I'd like to have a look on the supplementary materials before.

Minor comments

- in the first page "citation" reports Microorganisms instead of Diversity

- the reference list is not well formatted for Diversity and contains a lot of typesetting errors. Please, check.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf