Effect of the Added Acyl Homoserine Lactones on Separated Free-Living Marine Bacteria as a Model of Quorum Sensing

Round 1

Reviewer 1 Report

1. Title. The whole article discusses quorum sensing; experimental part does not describe marine bacteria immobilized in gels. So, the current title does not suit well. Please change the title for another one that will describe the presented materials more precise. For example: “Effect of the added acyl homoserine lactones on separated free-living marine bacteria as a model of quorum sensing”.

2. Abstract. The abstract contains i) speculation about quorum sensing in some sparse populations of marine bacteria, ii) just two lines about the marine bacteria response to AHLs. Please rewrite the abstract according to the presented results in other lay-out. As example: 1) Possible signaling molecules were collected from marine water, separated, and concentrated as some organic matter. Their identification with an Agrobacterium tumefaciens-assay indicated the presence of AHLs. 2) In fall 2002 and spring 2003, free-living (single cells) fraction of marine bacterioplankton was sampled and separated. Various AHLs were added to these disperse populations (10 ⁹ cells/L). The studied bacterial communities responded with change in the thymidine incorporation. 3) The results are discussed as existence of active reaction of marine free-living bacteria to signaling molecules even in sparse populations. Fact of bacterial response to signaling molecules in so sparse populations is of great significance for marine and environmental sciences.

 

3. Materials and Methods.

3.1. Lines 138-140. Both measurement of the tritiated thymidine into DNA and bacteria counting are not “Measurement of bacterial properties”. It will be better to present this paragraph as: i) “Bacterial number in marine water. Bacterial abundance was determined by direct count under microscope (magnification x…) on filters with acridine orange staining ([38])”. ii) to replace “Bacterial incorporation rates were determined by addition of tritiated thymidine ([36]; [37])” from the lines 138-139to the line 154, namely: after “…method of Smith and Azam ([37]).”

3.2. Lines 152-153. Please add temperature data “…incubated at near in situ temperature (??? ^oC in fall and … ^oC in spring) for 1 - 2 hr”.

 

4. Results and Discussion.

4.1. Please include subsection titles:

“3.1. Identification of origin of signaling molecules in the Galveston Bay water” for lines 157-186;

“3.2. Response of the sparse bacterial community to AHLs, November 2002” for lines 187-232;

“3.3. Response of the sparse bacterial community to AHLs, April 2003” for lines 233-309.

4.2.

Line 157: delete brackets.  

Lines 167-168: change “Previous work in estuarine and other marine systems has shown…” to “Previous works in estuarine and other marine systems have shown…”.

Line 173: to make your opinion clear for readers, change for “was composed of free-living i.e. non-aggregated, disperse bacteria”.

Lines 174-180: replace this text into “Materials and Methods”, subsection “experimental protocol”.

Lines 180-182: delete from “Although…” to “Thus”.

Lines 258-260: number (abundance) of bacteria, cells/L. Please add some information: i) was it counted in the whole marine waster sample or in the filtered one? ii) was in counted in April only or in November too?

 

5. Conclusions.

By my humble opinion, conclusions have to present not only some judgment but also a decision based on the paper’s results. In the current form, the presented conclusions are limited just with some debatable reasoning. I ask authors to rewrite conclusions adding some postulates, namely: the work showed that: i) marine water contained some signaling molecules which provoked bacterial reactions (at least - of A. tumefaciens), ii) at least, some of these agents were AHLs, iii) dispersed populations of filtered marine bacterioplankton showed response to addition of AHLs, iv) bacterial populations sampled in 6 months confirmed response but for other AHL (the reasons of the change are unknown: they may be related both with changes in population composition or in chemical composition of the water); v) the used concentrations of AHLs were high but the permitted to make this investigation as a model; vi) the main: in total, the presented work showed existence of response to signaling molecules in a highly dispersed bacterial community.

Comments for author File: Comments.pdf

Author Response

1. Title. The whole article discusses quorum sensing; experimental part does not describe marine bacteria immobilized in gels. So, the current title does not suit well. Please change the title for another one that will describe the presented materials more precise. For example: “Effect of the added acyl homoserine lactones on separated free-living marine bacteria as a model of quorum sensing”.

Answer: done

2. Abstract. The abstract contains i) speculation about quorum sensing in some sparse populations of marine bacteria, ii) just two lines about the marine bacteria response to AHLs. Please rewrite the abstract according to the presented results in other lay-out. As example: 1) Possible signaling molecules were collected from marine water, separated, and concentrated as some organic matter. Their identification with an Agrobacterium tumefaciens-assay indicated the presence of AHLs. 2) In fall 2002 and spring 2003, free-living (single cells) fraction of marine bacterioplankton was sampled and separated. Various AHLs were added to these disperse populations (10 ⁹ cells/L). The studied bacterial communities responded with change in the thymidine incorporation. 3) The results are discussed as existence of active reaction of marine free-living bacteria to signaling molecules even in sparse populations. Fact of bacterial response to signaling molecules in so sparse populations is of great significance for marine and environmental sciences.

 Answer: done

3. Materials and Methods.

3.1. Lines 138-140. Both measurement of the tritiated thymidine into DNA and bacteria counting are not “Measurement of bacterial properties”. It will be better to present this paragraph as: i) “Bacterial number in marine water. Bacterial abundance was determined by direct count under microscope (magnification x…) on filters with acridine orange staining ([38])”. ii) to replace “Bacterial incorporation rates were determined by addition of tritiated thymidine ([36]; [37])” from the lines 138-139to the line 154, namely: after “…method of Smith and Azam ([37]).”

3.2. Lines 152-153. Please add temperature data “…incubated at near in situ temperature (??? ^oC in fall and … ^oC in spring) for 1 - 2 hr”.

 Answer: done

4. Results and Discussion.

4.1. Please include subsection titles:

“3.1. Identification of origin of signaling molecules in the Galveston Bay water” for lines 157-186;

“3.2. Response of the sparse bacterial community to AHLs, November 2002” for lines 187-232;

“3.3. Response of the sparse bacterial community to AHLs, April 2003” for lines 233-309.

4.2.

Line 157: delete brackets. 

Lines 167-168: change “Previous work in estuarine and other marine systems has shown…” to “Previous works in estuarine and other marine systems have shown…”.

Line 173: to make your opinion clear for readers, change for “was composed of free-living i.e. non-aggregated, disperse bacteria”.

Lines 174-180: replace this text into “Materials and Methods”, subsection “experimental protocol”.

Lines 180-182: delete from “Although…” to “Thus”.

Lines 258-260: number (abundance) of bacteria, cells/L. Please add some information: i) was it counted in the whole marine waster sample or in the filtered one? ii) was in counted in April only or in November too?

 Answer: all done

5. Conclusions.

By my humble opinion, conclusions have to present not only some judgment but also a decision based on the paper’s results. In the current form, the presented conclusions are limited just with some debatable reasoning. I ask authors to rewrite conclusions adding some postulates, namely: the work showed that: i) marine water contained some signaling molecules which provoked bacterial reactions (at least - of A. tumefaciens), ii) at least, some of these agents were AHLs, iii) dispersed populations of filtered marine bacterioplankton showed response to addition of AHLs, iv) bacterial populations sampled in 6 months confirmed response but for other AHL (the reasons of the change are unknown: they may be related both with changes in population composition or in chemical composition of the water); v) the used concentrations of AHLs were high but the permitted to make this investigation as a model; vi) the main: in total, the presented work showed existence of response to signaling molecules in a highly dispersed bacterial community.

Answer: done

Author Response File: Author Response.pdf

Reviewer 2 Report

1.     This is a significant work where the authors suggest the presence of AHL in the Galveston water bay. This autoinducer molecule, related to the QS system, is known to accumulate as population density increases.  In this work, AHL in relation their interaction with the free-living community of the water, was studied by looking at the growth of bacteria as a measure of thymidine incorporation. The authors present their observations on differences in thymidine incorporation on free-living bacteria from Galveston Bay in November and April.

Although differences in the bacterial community composition at two different time is presumed to have contributed to their difference in the response upon AHL addition, it would be helpful to know if AHLs interact with other one or more QS bacteria, and how cell density can affect this response. Authors could add positive controls or cite works where a difference in thymidine incorporation could be observed in any QS organisms at a certain density. This might make it clear that the observed difference in response is due to community differences and no other factors. Since both Octanoyl and Dodecanoyl show intriguingly different responses at these two-time points, it would be nice to validate similar/different responses with a positive control to capture their interaction with the AHL.

And it would also be interesting if any previous works could be cited to show species composition changes between the two seasons (if available).

 

2.     There are multiple typos with “ ° C.”

 

3.     Section from Line 138 can be combined with the section starting at Line 141 for clarity.

n/a

Author Response

1. This is a significant work where the authors suggest the presence of AHL in the Galveston water bay. This autoinducer molecule, related to the QS system, is known to accumulate as population density increases.  In this work, AHL in relation their interaction with the free-living community of the water, was studied by looking at the growth of bacteria as a measure of thymidine incorporation. The authors present their observations on differences in thymidine incorporation on free-living bacteria from Galveston Bay in November and April.

Although differences in the bacterial community composition at two different time is presumed to have contributed to their difference in the response upon AHL addition, it would be helpful to know if AHLs interact with other one or more QS bacteria, and how cell density can affect this response. Authors could add positive controls or cite works where a difference in thymidine incorporation could be observed in any QS organisms at a certain density. This might make it clear that the observed difference in response is due to community differences and no other factors. Since both Octanoyl and Dodecanoyl show intriguingly different responses at these two-time points, it would be nice to validate similar/different responses with a positive control to capture their interaction with the AHL.

 

Answer:

 To repeat experiments is not possible. However, we put our experimental evidence in better perspective, and added more interpretations. “While there are newer methods and approaches to studying QS using AHLs our approach is robust for showing the presence of QS bacteria in the water sample. In the absence of a clearly positive response, however, our approach can be more ambiguous. In support of our explanation for the November results, Lin et al. (2003) showed that Vibrio vulnificus, a gram negative bacterium and AHL producer, shows near minimum abundance in the fall of 2000, corresponding to lower temperature and salinity of the water. For the spring/summer of 2001, they showed near maximum abundance. Furthermore, Santschi  [1995] showed that in general, phosphate concentrations in Galveston Bay waters are highest in spring/summer, while near a minimum in the fall/winter months. Total Nitrogen loadings of Galveston Bay are very high ([Santschi, 1995; Warnken and Santschi, 2009]), one of the highest of US estuaries, and most likely, not limiting during November 2002 and April 2003. The observed difference in response is thus likely due to community differences.

\Also, an explanation for the difference in Octanoyl and Dodecanoyl responses in was given in the manuscript. “These changes in the behavior of the natural bacterial community may be due to a change in community structure.  The mechanism for using quorum sensing between the two communities may be similar, however, with quorum sensing occurring as bacteria congregate about food sources, i.e., marine gels.  The response of the species in the April assemblage is entirely different from the response seen in the November community.  The responding bacteria in the April community may utilize AHLs to produce exoenzymes or to shift metabolism from cell maintenance to cell growth as the water becomes warmer and nutrients more plentiful.  As the responding bacteria are likely different species from the November responders (lower temperature and nutrients), they may utilize QS at a lower cell or molecule density as the gel is aggregating and thus shift to cell growth as nutrients become plentiful. Although these hypotheses are presently merely speculation, investigation of these and other hypotheses may give researchers important insights into the functions of free-living bacterial communities in marine ecosystems.”

 

And it would also be interesting if any previous works could be cited to show species composition changes between the two seasons (if available).

 

Answer: See explanations above about V. vulnificus differences between the fall and spring season.

 

2. There are multiple typos with “ ° C.”

 

Answer: fixed, thanks.

 

3. Section from Line 138 can be combined with the section starting at Line 141 for clarity.

 

Answer: We followed the recommendations of Reviewer 1

[3.1. Lines 138-140. Both measurement of the tritiated thymidine into DNA and bacteria counting are not “Measurement of bacterial properties”. It will be better to present this paragraph as: i) “Bacterial number in marine water. Bacterial abundance was determined by direct count under microscope (magnification x…) on filters with acridine orange staining ([38])”. ii) to replace “Bacterial incorporation rates were determined by addition of tritiated thymidine ([36]; [37])” from the lines 138-139to the line 154, namely: after “…method of Smith and Azam ([37]).”]

and revised this section accordingly.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The study done by Schultz and Santschi is insightful and thought-provoking. The research presented in this article is significant highlighting the processes controlled by quorum sensing and the extent to which it is utilized in free-living marine bacteria, and other free-living bacterial communities, which offers an exciting challenge for future endeavors. The authors have taken a rigorous approach to their research and have provided compelling evidence to support their findings and I believe that it would make a valuable contribution to the ongoing discourse in the field of marine ecosystems, however, I have some concerns about the methodology and conclusions presented in the research:

1.     The research has been conducted in late 2002 and the first of 2003, twenty years ago. While the research presented in the article was groundbreaking at the time, I have some concerns about the relevance and validity today, in other words, the methodology and data used in the study may have been appropriate for the time period in which the research was conducted, but may not meet current standards for scientific rigor and this might raise questions about the validity of the results and undermines the overall credibility of the research.  So, the authors are encouraged to repeat the experiment and compare the findings.

2.     Authors did not provide a proper explanation for the thymidine incorporation seen in this community of bacteria toward octanoyl AHL in the November study and its nonsignificant effect on thymidine incorporation, while both decanoyl and dodecanoyl stimulated an increase in incorporation in April one.

 

3.     Italicize all bacterial names.

Author Response

The study done by Schultz and Santschi is insightful and thought-provoking. The research presented in this article is significant highlighting the processes controlled by quorum sensing and the extent to which it is utilized in free-living marine bacteria, and other free-living bacterial communities, which offers an exciting challenge for future endeavors. The authors have taken a rigorous approach to their research and have provided compelling evidence to support their findings and I believe that it would make a valuable contribution to the ongoing discourse in the field of marine ecosystems, however, I have some concerns about the methodology and conclusions presented in the research:

1. The research has been conducted in late 2002 and the first of 2003, twenty years ago. While the research presented in the article was groundbreaking at the time, I have some concerns about the relevance and validity today, in other words, the methodology and data used in the study may have been appropriate for the time period in which the research was conducted, but may not meet current standards for scientific rigor and this might raise questions about the validity of the results and undermines the overall credibility of the research.  So, the authors are encouraged to repeat the experiment and compare the findings.

Answer: To repeat experiments is not possible. However, we put our experimental evidence in better perspective, and added more interpretations. “While there are newer methods and approaches to studying QS using AHLs our approach is robust for showing the presence of QS bacteria in the water sample. In the absence of a clearly positive response, however, our approach can be more ambiguous. In support of our explanation for the November results, Lin et al. (2003) showed that Vibrio vulnificus, a gram negative bacterium and AHL producer, shows near minimum abundance in the fall of 2000, corresponding to lower temperature and salinity of the water. For the spring/summer of 2001, they showed near maximum abundance. Furthermore, Santschi  [1995] showed that in general, phosphate concentrations in Galveston Bay waters are highest in spring/summer, while near a minimum in the fall/winter months. Total Nitrogen loadings of Galveston Bay are very high ([Santschi, 1995; Warnken and Santschi, 2009]), one of the highest of US estuaries, and most likely, not limiting during November 2002 and April 2003.

2. Authors did not provide a proper explanation for the thymidine incorporation seen in this community of bacteria toward octanoyl AHL in the November study and its nonsignificant effect on thymidine incorporation, while both decanoyl and dodecanoyl stimulated an increase in incorporation in April one.

Answer: An explanation was given in the manuscript. “These changes in the behavior of the natural bacterial community may be due to a change in community structure.  The mechanism for using quorum sensing between the two communities may be similar, however, with quorum sensing occurring as bacteria congregate about food sources, i.e., marine gels.  The response of the species in the April assemblage is entirely different from the response seen in the November community.  The responding bacteria in the April community may utilize AHLs to produce exoenzymes or to shift metabolism from cell maintenance to cell growth as the water becomes warmer and nutrients more plentiful.  As the responding bacteria are likely different species from the November responders (lower temperature and nutrients), they may utilize QS at a lower cell or molecule density as the gel is aggregating and thus shift to cell growth as nutrients become plentiful. Although these hypotheses are presently merely speculation, investigation of these and other hypotheses may give researchers important insights into the functions of free-living bacterial communities in marine ecosystems.”

3. Italicize all bacterial names.

Answer: Done

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

All the comments have been addressed. The manuscript can be accepted with these changes to the abstract and discussion.  

 

I have one minor comment. Please make the section heading 2.3 more specific than writing Experimental protocol.

Reviewer 3 Report

All comments are properly addressed