Filamentous phages reduce bacterial growth in low salinities

Being non-lytic, filamentous phages can replicate at high frequencies and often carry virulence factors, which are important in the evolution and emergence of novel pathogens. However, their net effect on bacterial fitness remains unknown. To understand the ecology and evolution between filamentous phages and their hosts, it is important to assess (i) fitness effects of filamentous phages on their hosts and (ii) how these effects depend on the environment. To determine how the net effect on bacterial fitness by filamentous phages changes across environments, we constructed phage–bacteria infection networks at ambient 15 practical salinity units (PSU) and stressful salinities (11 and 7 PSU) using the marine bacterium, Vibrio alginolyticus and its derived filamentous phages as model system. We observed no significant difference in network structure at 15 and 11 PSU. However, at 7 PSU phages significantly reduced bacterial growth changing network structure. This pattern was mainly driven by a significant increase in bacterial susceptibility. Our findings suggest that filamentous phages decrease bacterial growth, an indirect measure of fitness in stressful environmental conditions, which might impact bacterial communities, alter horizontal gene transfer events and possibly favour the emergence of novel pathogens in environmental Vibrios.


Accept with minor revision (please list in comments)
Comments to the Author(s) I enjoyed reading this paper. An understanding of how abiotic factors influence bacteria-phage networks is one of fundamental and applied interest. I have a couple of minor suggestions: I am glad that the authors have pointed out that studying the interactions over a longer (evolutionary time-scales) is required. One could predict that bacteria will adapt to counter the negative effects on fitness at lower salinities. With this point in mind I think the authors should scale back or tone down the language relating to the potential consequences of this reduced salinity in the future with respect to the evolution and emergence of pathogens i.e. last sentence of the abstract and Line 298-299 e.g. "we can expect the emergence of new pathogenic Vibrios in response to decreasing salinities." The use of the word "can" expect sounds as though this outcome is almost guaranteed… Similarly, I think the final sentence of the abstract is overstated based on the actual findings presented.

Spell out PSU in the first instance Line 181 check font
Decision letter (RSOS-191669.R0) 18-Oct-2019 Dear Dr Wendling On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-191669 entitled "Filamentous phages reduce bacterial growth in low salinities" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) In this paper, the authors test phage host range under varying salinities, using conditions that represent possible variation during regular fluctuations as well as under climate change. Overall, this is a well-written paper with interesting results. I have a few minor questions/concerns listed below: Minor concerns: 1. Depending on the journal space requirements, I would recommend moving more (or all) of the supplementary methods into the main text. Similarly, I would recommend restating more of the essential method details that are described in other papers (e.g. that phages were originally induced with Mitomycin C and diluted in TM buffer). This would be more convenient for the reader.
2. How reliable is the growth rate reduction assay at all salinities? Is it possible that phages are more harmful at low salinity but susceptibility is actually not higher? I.e. could you have cryptic infections that do not infect at high rates or do not infect with high virulence unless at low salinity? One possibility is that the stress from the low salinity leads to a higher rate of spontaneous phage induction, and that phages do infect at 15 PSU but do so without causing significant loss of host growth. If possible, I would suggest doing PCR assays on a random subsample of both negative and positive results to confirm the growth curve assay inferences.
3. Because the 7 PSU treatments grew slower, the authors varied the timing of the infection assay to try to do infections at the same relative part of the bacterial growth curves. Looking at the supplementary figure with bacterial growth curves, low salinity not only had slower growth but prolonged growth (and in some graphs what appears to be diauxic growth). Is it possible that these changes in timing, though, might explain some of the differences in bacterial susceptibility? That is to say, might low salinity increase the time window for phage infection? 4. Very minor semantics issue: In figure S6, a label refers to "number of phages causing lytic infections." The use of "lytic" here seems inappropriate since the assay does not distinguish between reduced growth and increased mortality (and since the phages are filamentous).

Reviewer: 2
Comments to the Author(s) I enjoyed reading this paper. An understanding of how abiotic factors influence bacteria-phage networks is one of fundamental and applied interest. I have a couple of minor suggestions: I am glad that the authors have pointed out that studying the interactions over a longer (evolutionary time-scales) is required. One could predict that bacteria will adapt to counter the negative effects on fitness at lower salinities. With this point in mind I think the authors should scale back or tone down the language relating to the potential consequences of this reduced salinity in the future with respect to the evolution and emergence of pathogens i.e. last sentence of the abstract and Line 298-299 e.g. "we can expect the emergence of new pathogenic Vibrios in response to decreasing salinities." The use of the word "can" expect sounds as though this outcome is almost guaranteed… Similarly, I think the final sentence of the abstract is overstated based on the actual findings presented. Decision letter (RSOS-191669.R1)

13-Nov-2019
Dear Dr Wendling, It is a pleasure to accept your manuscript entitled "Filamentous phages reduce bacterial growth in low salinities" in its current form for publication in Royal Society Open Science. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter.
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Please see the Royal Society Publishing guidance on how you may share your accepted author manuscript at https://royalsociety.org/journals/ethics-policies/media-embargo/. We are grateful for the valuable reviewer comments, which we think improved the manuscript considerably. Accordingly, we included a new data set demonstrating, that resident phages do not produce more phages, which might be costly for their host, at low salinities. This confirms our hypothesis, that indeed infecting phages are causing the strong reduction in bacterial growth at low salinities. Please find our point by point answers below.
Thank you for considering our manuscript for publication.

Yours sincerely, Carolin Wendling
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) In this paper, the authors test phage host range under varying salinities, using conditions that represent possible variation during regular fluctuations as well as under climate change. Overall, this is a well-written paper with interesting results. I have a few minor questions/concerns listed below: Minor concerns: 1. Depending on the journal space requirements, I would recommend moving more (or all) of the supplementary methods into the main text. Similarly, I would recommend restating more of the essential method details that are described in other papers (e.g. that phages were originally induced with Mitomycin C and diluted in TM buffer). This would be more convenient for the reader.
Response: We now moved all the sections describing the methods from the ESM to the main document and include an additional section on phage extraction. We cite Wendling, Goehlich, Roth (2018) for details about phage extraction. Neither in that nor in the present study did we use mitomycin C. We simply purified the supernatant from exponentially growing bacteria, which we already mentioned in the manuscript. As suggested, we now mention that phages were diluted in TM-buffer.
2. How reliable is the growth rate reduction assay at all salinities? Is it possible that phages are more harmful at low salinity but susceptibility is actually not higher? I.e. could you have cryptic infections that do not infect at high rates or do not infect with high virulence unless at low salinity? One possibility is that the stress from the low salinity leads to a higher rate of spontaneous phage induction, and that phages do infect at 15 PSU but do so without causing significant loss of host growth. If possible, I would suggest doing PCR assays on a random subsample of both negative and positive results to confirm the growth curve assay inferences.
Response: As these are filamentous phages, phage induction as for instance described for head-tail prophages does not happen. We can thus exclude that induction sensu stricto differs between salinities, simply because filamentous phages can't be induced. However, it is possible that phage production differs between salinities. We thus now measured the production of resident filamentous phages for each strain and found no difference among salinities. We now say so in the manuscript, lines 164-195, lines 228-231, and lines 275 -280.