Living in a Puddle of Mud: Isolation and Characterization of Two Novel Caulobacteraceae Strains Brevundimonas pondensis sp. nov. and Brevundimonas goettingensis sp. nov.

Round 1

Reviewer 1 Report

The aim of this paper was isolation and characterisation of bacterial strain of the Caulobacteracea family. These strains can be used in further investigation as a host system to access the associated phage diversity present in the water-related environments. Twelve different sampling sites were collected: 1) frog’s lettuce (Groenlandia densa) (PM), 2) pond water (PW), 3) surface water near pond algae (WSA), 4) surface water near frog’s lettuce (WSP), 5) surface water of reed (WSR) and 6) surface water close from Weende River entrance (WSW), Weende River nearby the oligotrophic pond 7) (RW) and (mixed = different sizes) river stones 8-9) (RS and MRS), 10) eutrophic pond surface water (POW) and 11) surface water of stale eutrophic pond (PSW), 12) samples from a puddle close by the eutrophic pond (PUW). Cultures inoculated with oligotrophic samples revealed a similar composition of the microbial community irrespective of the sampling site, while eutrophic water samples show more diverse bacterial communities.

The study describes two novel species of the genus Brevundimonas (LVF1T and LVF2T). Both novel species were characterized genomically, morphologically, and physiologically. Physiological characterization revealed that both strains grow optimal at 30 °C, showing minor differences for tested metabolic activities but resistance potential with respect to medically relevant antibiotics.

The article makes an important contribution to the microbiology of the freshwater environment and potential host systems for studying phage diversity in the environment. It is written thoroughly, correctly, and professionally.

I only have a few suggestions for technical correction.

Specific comments:

Line 81-82 Could you please describe the location of the eutrophic pond in more detail?

Line 293-297 Can the authors present the physico-chemical characteristics of different water samples? It would be interesting to make comparisons between them.

Line 523-524 Can the authors offer an explanation for the fact that no members of Caulobacterales were found in the WSW and WSR samples?

Author Response

Specific comments:

Line 81-82 Could you please describe the location of the eutrophic pond in more detail?

Response: Additional information was introduced in new line 85 and coordinates are already provided in Table 1

Line 293-297 Can the authors present the physico-chemical characteristics of different water samples? It would be interesting to make comparisons between them.

Response: We cannot supply additional physico-chemical characteristics of the different water samples. The remains of the samples are not enough to gather these data, but we suggested this with respect to content of different antibiotics (new lines 592-593).

Line 523-524 Can the authors offer an explanation for the fact that no members of Caulobacterales were found in the WSW and WSR samples?

Response: Additional information was introduced in new lines 536-540.

Reviewer 2 Report

General Comments

In the manuscript “Living in a puddle of mud: Isolation and characterization of two novel Caulobacteraceae strains Brevundimonas pondensis sp. nov. and Brevundimonas goettingensis sp. nov.”, Friedrich and co-authors describe the isolation and characterization of two strains of Caulobacteraceae isolated from different compartments of a eutrophic and an oligotrophic pond in Germany. The manuscript mentions several criteria of interest to distinguish newly isolated microbial strains, such as morphological, physiological, and genomic characteristics. A lot of technical information is given in the Materials and Methods, Results and Supplementary sections, which is especially important for reproducibility purposes. While the manuscript has a lot of potential, several drawbacks should be addressed before being considered for publication.  

First, while this is a simple adjustment, references should be numbered. They are cited as numbers in the text, but those numbers are absent from the list of references, so it is cumbersome to figure out what a given statement is referring to.

Furthermore, the main goal of the study is unclear. The authors mention that their aim is to “isolate and characterize a bacterial strain of the Caulobacteracea family to serve in further studies as a host system to access the viral diversity of Caulobacteracea-related phages present in the environment”. Why is this important? The reader should not have to figure it out by themselves. Adding a sentence or two should be sufficient.

Another issue is that the two strains were named like new species rather than new strains. The two selected isolates were more than 99% identical to other Brevundimonas species on the basis of their 16S rRNA, so they are of the same species as other Brevundimonas. This is mentioned in the title, so the authors are clearly aware of this aspect. Therefore, the strains should not be named Brevundimonas pondensis/goettingensis sp. nov., but Brevundimonas sp. strain pondensis/goettigensis. A more thorough analysis of systematics should be done to make sure that this is correct, but the name of the strains must be adjusted according to current nomenclature norms.

While the phylogenetic analysis is crucial for the identification and characterization of both new isolates and has been an essential inclusion in this manuscript, it would have been important to include bacterial genera that are phylogenetically really close to the isolates. A quick analysis showed that the full 16S rRNA gene of the Brevundimonas sp. LVF2 strain (from Table S5) is >98% identical to several Caulobacter sp. strains. The best hits were indeed Brevundimonas spp., but considering how common misclassifications are in bacterial systematics (e.g. with the Escherichia and Shigella genera), it would be reasonable to include an additional species or two in the ANI analysis. Perhaps the strains could be Caulobacter sp. after all.

Lastly, the discussion could be improved. As was mentioned about the introduction, the discussion also lacks a clear goal, and a more in-depth analysis of the topic is necessary. Most results are barely discussed or are simply repeating what was said in the Results section.  

In short, the manuscript has potential but the data, including the sequenced genomes themselves, should be analyzed and interpreted more thoroughly, as the manuscript is mainly descriptive, and the current naming scheme should be revised.

Specific Comments

1. Throughout the text: References are cited as numbers in the text, but there are no numbers in the References section. Those numbers should be added.
2. Line 44: I recommend reading this paper by Wilhelm RC in 2018: https://0-doi-org.brum.beds.ac.uk/10.1038/s41396-018-0257-z. It shows that Caulobacter is typically more abundant in terrestrial environments, so this paradigm has been challenged recently.
3. Line 64: no comma is required in this sentence, i.e. should be written as "It infects both prosthecate...“
4. Lines 122-124: Was the sequencing performed in-house or by an external company? Please add this information for clear backtracking.
5. Line 134: the in-text reference has a different formatting here.
6. Line 142: Please mention amplicon sequence “variant” before using the abbreviation “ASV”. Furthermore, it would be best to mention at least that the representative sequences were assigned to ASVs and then that the filtered reads were mapped to those representative sequences (ASVs). There seems to be a gap in the protocol in this section.
7. Line 291: What is meant by the “from the resistances”?
8. Line 303: “There” was also no significant…
9. Line 305: “led” instead of “lead”. What is meant by “more diverse”? Was there a higher number of orders (richness), or was it more even, was some diversity index higher than in oligotrophic samples? This is also valid for line 313 and for the statement “relatively homogenous”.
10. Line 307: as “the” most promising
11. Lines 333-337: Please reword those sentences for clarity
12. Lines 508-509: Is there any reference to this claim? Acquiring or losing resistance genes is not necessarily a proof that two isolates are from different species
13. The authors rely a lot on metabolic potential to compare LVF1 and LVF2 to previously characterized strains. However, many of those characteristics are missing in Table 3 for strains from other studies. Those strains should have been grown for this experiment and tested as well or parameters that are absent in at least one strain should be removed from the table, otherwise it is hard to truly compare them on this basis.
14. Line 558: no “s” in extreme
15. Line 571-573: I cannot find the reference because the list of references is not numbered, so I am not certain what it is supposed to prove. Environmental microbes such as Streptomyces and various species of mold produce medically relevant antibiotics. The fact that environmental Caulobacteraceae carry antibiotic resistance genes is not necessarily caused by environmental antibiotic contamination. A much deeper analysis of the samples would have been necessary to determine whether there could be antibiotic contamination at those sampling sites.
16. Throughout the text, words like “obvious” and “surprising” should be avoided. They have a subjective meaning.
17. Throughout the text, be careful about the use of “similarity” and “identity”, they are not interchangeable in the context of genomics.

Author Response

First, while this is a simple adjustment, references should be numbered. They are cited as numbers in the text, but those numbers are absent from the list of references, so it is cumbersome to figure out what a given statement is referring to.

Response: We apologize for this inconvenience. All references are now numbered.

Furthermore, the main goal of the study is unclear. The authors mention that their aim is to “isolate and characterize a bacterial strain of the Caulobacteracea family to serve in further studies as a host system to access the viral diversity of Caulobacteracea-related phages present in the environment”. Why is this important? The reader should not have to figure it out by themselves. Adding a sentence or two should be sufficient.

Response: Additional information was introduced in new lines 68–74.

Another issue is that the two strains were named like new species rather than new strains. The two selected isolates were more than 99% identical to other Brevundimonas species on the basis of their 16S rRNA, so they are of the same species as other Brevundimonas. This is mentioned in the title, so the authors are clearly aware of this aspect. Therefore, the strains should not be named Brevundimonas pondensis/goettingensis sp. nov., but Brevundimonas sp. strain pondensis/goettigensis. A more thorough analysis of systematics should be done to make sure that this is correct, but the name of the strains must be adjusted according to current nomenclature norms.

Response: Dear Reviewer, the isolated and here presented strains are the first representatives of two new species. You are right, on the 16S rRNA gene sequence they do not significantly differ from already known strains. However, it is known from other examples that taxonomic classification based only on this gene has limitations with respect to species separation and provides a good but by far not  perfect phylogenetic assignment. The most prominent examples are many species of the genus Bacillus, which cannot be separated from each other based solely on 16S rRNA gene sequences. To overcome this shortage whole-genome comparison between known and new strains can be used to determine the uniqueness of the strain. How particularly this is applied is described in Chun et al. 2018 (https://0-doi-org.brum.beds.ac.uk/10.1099/ijsem.0.002516). We followed the workflow by using the ANI algorithm for our whole genome sequence OGRI. As mentioned in manuscript, the ANI of both isolates are lower than 95% and therefore new species.Additionally, our isolates were confirmed as new species by the independent Type Strain Genome Server (TYGS) provided by the German Collection of Microorganisms and Cell Cultures GmbH DSMZ (https://tygs.dsmz.de/user_results/show?guid=e9c66d86-1436-41af-9542-c400641a5b3d). Information regarding that analysis was added to the manuscript in new lines 465–466.

While the phylogenetic analysis is crucial for the identification and characterization of both new isolates and has been an essential inclusion in this manuscript, it would have been important to include bacterial genera that are phylogenetically really close to the isolates. A quick analysis showed that the full 16S rRNA gene of the Brevundimonas sp. LVF2 strain (from Table S5) is >98% identical to several Caulobacter sp. strains. The best hits were indeed Brevundimonas spp., but considering how common misclassifications are in bacterial systematics (e.g. with the Escherichia and Shigella genera), it would be reasonable to include an additional species or two in the ANI analysis. Perhaps the strains could be Caulobacter sp. after all.

Response: Dear Reviewer, we agree that it is a good idea to include close related strains to the ANI analysis and those not necessarily classified as related species. However, as mentioned previously 16S rRNA gene-based classification is sometimes not the best choice to discriminate between species and in the same manner not to find the closest relative. To precisely identify the genome of the closest relative to our strain, we would need to align them to all available potential Caulobacteraceae. To address these challenges systematically, we submitted our strain to the Type Strain Genome Server (TYGS) provided by the German Collection of Microorganisms and Cell Cultures GmbH DSMZ (https://tygs.dsmz.de/user_results/show?guid=e9c66d86-1436-41af-9542-c400641a5b3d). The TYGS system extracts the 16S rRNA gene from the submitted genomes and identifies the closest related species via BLASTn. Next, all type strains genomes of the identified species are aligned to the genome of interest. Based on this, the pipeline assigns the isolates of interest as new strains of a known species or as representatives of a new species. This additional analysis confirmed the results presented by us and excluded the possibility that the new strains could be Caulobacter sp. after all. In order to avoid the obviously present ambiguities in the present manuscript, we included the information obtained through the TYGS system in the manuscript. For the sake of completeness, the ANI analysis (Figure 2) was complimented with the type strains suggested by TYGS.

Lastly, the discussion could be improved. As was mentioned about the introduction, the discussion also lacks a clear goal, and a more in-depth analysis of the topic is necessary. Most results are barely discussed or are simply repeating what was said in the Results section.  

Response: We stated our intentions in the introduction and tried to present them more clearly through this revision. We reworked the discussion to remove potential redundancies and make it more precise on the subject. We introduced an additional paragraph addressing the overall value of our results and their benefits to our scientific intentions and the value for the scientific community. We hope we have addressed your comments and have been able to improve our discussion to the required extent.

In short, the manuscript has potential but the data, including the sequenced genomes themselves, should be analyzed and interpreted more thoroughly, as the manuscript is mainly descriptive, and the current naming scheme should be revised.

Response: Dear Reviewer, thank you for the indication to be more precise with our statement. We hope we could improve the manuscript by working through the points above. Regarding the names, we hope we could convince you that our isolates are new species, which makes it legit to us to name them as previously done.

Specific Comments

1. Throughout the text: References are cited as numbers in the text, but there are no numbers in the References section. Those numbers should be added.

Response: Done as recommended

2. Line 44: I recommend reading this paper by Wilhelm RC in 2018: https://0-doi-org.brum.beds.ac.uk/10.1038/s41396-018-0257-z. It shows that Caulobacter is typically more abundant in terrestrial environments, so this paradigm has been challenged recently.

Response: Thank you for the hint to this manuscript. We implemented the  reference and the statement in the introduction.

3. Line 64: no comma is required in this sentence, i.e. should be written as "It infects both prosthecate...“

Response: Done as recommended

4. Lines 122-124: Was the sequencing performed in-house or by an external company? Please add this information for clear backtracking.

Response: Done as recommended

5. Line 134: the in-text reference has a different formatting here.

Response: Corrected as recommended

6. Line 142: Please mention amplicon sequence “variant” before using the abbreviation “ASV”. Furthermore, it would be best to mention at least that the representative sequences were assigned to ASVs and then that the filtered reads were mapped to those representative sequences (ASVs). There seems to be a gap in the protocol in this section.

Response: Done as recommended.

7. Line 291: What is meant by the “from the resistances”?

Response: Unfortunately, this was a copy error. It is now corrected.

8. Line 303: “There” was also no significant…

Response: Addressed as recommended

9. Line 305: “led” instead of “lead”. What is meant by “more diverse”? Was there a higher number of orders (richness), or was it more even, was some diversity index higher than in oligotrophic samples? This is also valid for line 313 and for the statement “relatively homogenous”.

Response: “Lead” was changed to “led”. We implemented further information in new lines 314–316 and 323–325.

10. Line 307: as “the” most promising

Response: Done as recommended.

11. Lines 333-337: Please reword those sentences for clarity

Response: Done as recommended.

12. Lines 508-509: Is there any reference to this claim? Acquiring or losing resistance genes is not necessarily a proof that two isolates are from different species

Response: We agree, and removed the respective statement from the manuscript.

13. The authors rely a lot on metabolic potential to compare LVF1 and LVF2 to previously characterized strains. However, many of those characteristics are missing in Table 3 for strains from other studies. Those strains should have been grown for this experiment and tested as well or parameters that are absent in at least one strain should be removed from the table, otherwise it is hard to truly compare them on this basis.

Response: Dear Reviewer, we fully agree that a direct comparison to our isolates to known and well-characterized strains would be great, but the focus of this investigation was on our isolates, which we intend to characterize as good as possible. With all respect, but reducing our results to the minimal available extend present in literature seems inappropriate to us. If doing so, we would limit our manuscript and also the research of others to whom such data are of interest. Thus, we we want to keep the Table in its present form.

14. Line 558: no “s” in extreme

Response: Corrected as recommended.

15. Line 571-573: I cannot find the reference because the list of references is not numbered, so I am not certain what it is supposed to prove. Environmental microbes such as Streptomyces and various species of mold produce medically relevant antibiotics. The fact that environmental Caulobacteraceae carry antibiotic resistance genes is not necessarily caused by environmental antibiotic contamination. A much deeper analysis of the samples would have been necessary to determine whether there could be antibiotic contamination at those sampling sites.

Response: We agree, and therefore we removed the respective statement from the manuscript.

16. Throughout the text, words like “obvious” and “surprising” should be avoided. They have a subjective meaning.

Response: Done as recommended.

17. Throughout the text, be careful about the use of “similarity” and “identity”, they are not interchangeable in the context of genomics.

Response: Checked as recommended.

Round 2

Reviewer 2 Report

Globally, I am satisfied with the revised manuscript as well as the authors’ response to my concerns. A few typographical errors were introduced in the discussion (see below), but aside from that, I believe that the manuscript is suitable for publication.

Lines 556-559: The end of the sentence is missing, so please correct this.

Line 566: “Physiological analyses” and “optimally”

Line 607: “particle-packed” would improve readability

Line 613: Remove “obtain”

Author Response

Lines 556-559: The end of the sentence is missing, so please correct this.

Response: Changed as recommended

Line 566: “Physiological analyses” and “optimally”

Response: Changed as recommended

Line 607: “particle-packed” would improve readability

Response: Changed as recommended

Line 613: Remove “obtain”

Response: Changed as recommended