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The minor concerns raised by the reviewers have been addressed satisfactorily.
The manuscript has been reviewed by three experts in the field, and all three considered the manuscript to be of high quality. The reviewers only had minor comments on the manuscript, mostly relating to presentation, though one reviewer also requests clarification on the motility assays and intracellular bacteria in the co-culture assays. Please revise your manuscript accordingly. I shall look forward to seeing the revised version.
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This manuscript describes the toxicity of pyrrolnitrin (PRN), phenazine (PHZ) and hydrogen cyanide (HCN) produced by Pseudomonas chlororaphis on the amoeba, Acanthamoeba castellanii. The results show that all 3 secondary metabolites inhibit and/or repel amoeba in vitro and that the expression of secondary metabolites increases in the presence of amoeba trophozoites but not supernatants from amoeba cultures.
The manuscript is well-written and clear and there are sufficient references in the background. All the figures are necessary and well presented. While the inhibition of predators by secondary metabolites have been shown previously, the bacterial strain used here has not been explored previously with respect to predation inhibition.
For the co-culture assays, it states that extracellular bacteria were counted (line 107-108) but what about intracellular bacterial numbers. In some cases bacteria can multiply inside the amoeba and you would not capture this by only plating extracellular cells. Have you considered lysing the amoeba and doing total bacterial counts?
What was the setup for the motility experiments? Were the amoeba added to the swim plate and if so how?
Specific comments
Line 130 – To maintain consistency the heading should be sentence case (e.g. Chemotaxis assay)
Lines 13 and 307 and – there should be a space between the number and unit (e.g. 72 h)
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Very interesting story and very excellent English. I learned a lot from it. However, just minor faults :1) in background introduction, I think the regulation of antibiotics production might not be necessary for this study. So I suggested this part is deleted (Line 40-46); 2) most references are listed with the full name of journal, but some are not (Line 425, 457, and 466).
The manuscript is well written and care has been taken to explain key concepts to the reader. Background to the study provides clear context and concisely conveys the current level of understanding in the field and the knowledge gap to be addressed. All figures are of a high quality and accompanied by detailed legends. All raw data has been supplied and is in an understandable format.
The study aims to determine if PA23 metabolites facilitate survival in the presence of a protozoan predator. It also aims to determine how these metabolites potentially protect the bacterium which produces them. As such, the study has a clear and well-defined research question.
Experiments are well conceived and well controlled. I commend the authors for the use of appropriate controls throughout their study. The number of replicates and experimental repeats performed allow for robust statistical analysis of the data.
Methods are detailed and could be easily reproduced by another researcher. For some experiments, the number of replicates are missing (see specific comments below). The methods employed are appropriate.
Statistical analyses have been applied where appropriate. The significance level of all analyses have been stated and provided on all figures. However, I am not 100% convinced regarding the appropriateness of a 2-way ANOVA for some of the analyses. Results are well described and easy to follow. Conclusions drawn are justified based on a solid analysis of the results. Potential anomalies are well explained. For example, potentially unexpected results for the PHZ- which can be explained by elevated levels of PRN produced by this strain.
Overall, I commend the authors for an interesting study investigating the mechanisms by which bacteria protect themselves from potential predators. I only have a few minor comments which are listed below:
Line 76, the abbreviated version of Escherichia coli (E. coli) is used prior to the full name being provided.
Line 212, “Conversely, trophozoite numbers were significantly elevated when grown on the PRN- and HCN- strains (Fig. 1)”. I find this description confusing; it implies that trophozoite numbers increased compared to Day 0. A more specific statement such as “trophozoite numbers were significantly higher when grown on PRN- and HCN- strains compared to those grown on the PHZ- strain at day 15.
Figure 2: I would find a line chart easier to view with regards to changes of the viability for each mutant over time. Is there a specific reason the authors chose to represent these data with a histogram?
In the methods section, a number of experiments are missing details on the number of replicates and repeats performed. These experiments are the “P. chlororaphis PA23 – Ac co-culture assays” “Chemotaxis assays “ and “ analysis of transcriptional fusions”
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