Elucidating the Capacity and Mechanism of Lactiplantibacillus plantarum in Synthesizing Essential Amino Acids from Non-Essential Amino Acids in a Novel Severely Deficient Medium

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes an important study in the understanding of the gut microbiota's role in synthesizing essential amino acids (EAAs). The development of an artificial medium that supports the growth of Lactiplantibacillus plantarum and the systematic depletion of EAAs to identify critical amino acids for survival and proliferation are novel approaches. The data are presented clearly and provides a strong foundation for future research to explore the role of the gut microbiota as a potential source of EAAs in host animals. The manuscript is well-written and presents novel and significant findings. Other comments are shown below:

1.Table 1, the isolation sources of the strains should be provided.

2, Figure 4, extracellular AA concentrations should be also standardized by the number of cells, just like those of the intracellular concentrations.

Author Response

We appreciate the reviewer's valuable comments, which have helped us significantly improve the paper. As indicated in the following responses, we have considered all these comments and suggestions in the revised version of our paper. We also improved the Table1 and added supplemental figures 3 and text to the revised manuscript. Our collective response to the reviewer to explain *point-by-point* the details of the revisions in the manuscript and our responses to the reviewer's comments are as follows:

 

Comment 1: Table 1, the isolation sources of the strains should be provided.

 

Our response: The isolation sources of the strains have been provided in Table 1 as requested (Line 111). We obtained all information of bacterial strain from NBRC and ATCC but some strains (ATCC 8014 and ATCC 7469) isolation source are unclear.

 

Comment 2: Figure 4, extracellular AA concentrations should be also standardized by the number of cells, just like those of the intracellular concentrations.

 

Our response: We appreciated the reviewers comment on improving the manuscript for readers. Standardizing extracellular amino acid (AA) concentrations by cell number is added in supplemental figure S3, modified lines 241-243, and added text for explanation in lines 270-272. In the main manuscript, we choose to represent the total concentration available in the culture medium in this study because the primary objective of the study being to evaluate the presence of essential AAs which removed from the medium after 48 hours of culture.  Unlike intracellular AAs, which directly reflect cellular metabolic activity and require normalization by cell count, extracellular AAs are determined by the combined dynamics of secretion and consumption by L. plantarum through the cultivation. We believe normalizing by culture volume ensures a consistent and reliable metric for comparing extracellular AA levels. We also added the results that were standardized by the number of cells in the supplementary figure 3 to support understanding of the results for readers.

Reviewer 2 Report

Comments and Suggestions for Authors

1. The first paragraph of the introduction part should be reorganized into 2-3 paragraphs for better readability.

2. Fig 6G could be improved for better understanding.

3. Could some of the data shown in the figs (e.g. fig 3 (c-f))  be transferred to tables?

4. Please confirm the proper usage of the abbreviations. e.g. line 135 'lactic acid bacteria', but you have mentioned 'LAB' as the abbreviation before. 

Author Response

Responses to Reviewer 2

 

We appreciate the reviewer's valuable comments, which have helped us significantly improve the paper. As indicated in the following responses, we have considered all these comments and suggestions in the revised version of our paper. We also improved the paragraph, Figure 6G, corrected the abbreviation, and tried to transfer some figure to table. Our collective response to the reviewer to explain *point-by-point* the details of the revisions in the manuscript and our responses to the reviewer's comments are as follows:

 

Comment 1: The first paragraph of the introduction part should be reorganized into 2-3 paragraphs for better readability.

 

Our response: Thank you for your suggestion. In the first section of the introduction, we have divided it into two paragraphs as per your suggestion (Line 38-39).

 

Comment 2: Fig 6G could be improved for better understanding.

 

Our response: Thank you for your comment. We have revised Figure 6G to clearly show the consumption and secretion of cysteine without the production of methionine, based on our experimental results. This revision makes the figure easier to understand and ensures it aligns with the data presented in the study (Line 312). We have also added some sentences in figure 6G legend (Lines 327 to 329).

 

Comment 3: Could some of the data shown in the figs (e.g. fig 3 (c-f)) be transferred to tables?

 

Our response: Thank you for the suggestion. We have tried to transfer figure 3c, 3d, and 3I as follows:

Data relates to Figure 3

Assay	Strains	Incubation time (hour)
		0h		24h		48h		72h
		Mean	SD	Mean	SD	Mean	SD	Mean	SD
△ Lysine	Strain 1	0.103	0.01	1.567	0.05	1.574	0.1	1.54	0.11
	Strain 2	0.102	0.01	0.203	0.01	0.231	0.02	0.247	0.02
	Strain 3	0.101	0	0.376	0.06	0.82	0.02	0.84	0.02
	Strain 4	0.097	0	0.205	0.01	0.232	0.02	0.248	0.02
	Strain 5	0.103	0	0.195	0.01	0.232	0.01	1.062	0.11
△ Threonine	Strain 1	0.103	0.01	1.574	0.03	1.546	0.07	1.532	0.11
	Strain 2	0.102	0.01	0.932	0.15	1.038	0.21	1.053	0.2
	Strain 3	0.101	0	0.395	0.06	1.069	0.13	1.124	0.15
	Strain 4	0.097	0	0.548	0.04	1.368	0.05	1.419	0.03
	Strain 5	0.103	0	0.751	0.11	1.278	0.13	1.429	0.24
△ Lysine and △ Threonine	Strain 1	0.1	0	1.5	0.03	1.47	0.08	1.45	0.04
	Strain 2	0.1	0	0.9	0.1	0.92	0.16	0.93	0.16
	Strain 3	0.11	0	0.28	0.03	0.87	0.07	1.02	0.05
	Strain 4	0.1	0	0.32	0.03	1.1	0.11	1.37	0.08
	Strain 5	0.1	0	0.28	0.06	0.82	0.13	0.93	0.03
The effect of single and double EAA omission on the growth of LAB strains in an artificial medium. Changes in OD 570 nm of each bacteria strain in media with single and double EAA omissions for culturing over the indicated time. Strain 1: Lactiplantibacillus plantarum (ATCC 8014); Strain 2: Lactobacillus delbrueckii (ATCC 9649); Strain 3: Lactocaseibacillus rhamnosus (ATCC 7469a); Strain 4: Lactocaseibacillus casei (ATCC 393); and Strain 5: Lactocaseibacillus paracasei subsp. Paracasei (ATCC 25302). The symbol △ indicates that a specific EAA was omitted from the artificial medium. Data represent the mean ± SD from 3 independent experiments performed in triplicate.

As shown in this Table, the information of exact values could be healpful for readers but numbers of data are too much even in 3 figures. In addition, our data comprises following key components: the growth curves of LAB strains and the dynamics of amino acid consumption and secretion. For the growth curves, we believed presenting the data as figures is easy to capture the temporal progression of bacterial growth, providing a visual correlation between time and optical optical density. These visual changes are helpful for understanding how LAB strains grow under specific conditions and enable readers to identify trends, such as increases, decreases, more easily than those shown by Table.

 

Hence, we believe that figures are the most effective way to communicate these complex data and facilitate a deeper understanding of the metabolic functions of LAB strains.

 

Comment 4: Please confirm the proper usage of the abbreviations. e.g. line 135 'lactic acid bacteria', but you have mentioned 'LAB' as the abbreviation before.

 

Our response: Thank you for pointing this out. We have revised the text to ensure consistent use of the abbreviation 'LAB' for 'lactic acid bacteria' throughout the manuscript (Line 138, 169, 192).