Reconstructing the transcriptional regulatory network of probiotic L. reuteri is enabled by transcriptomics and machine learning

ABSTRACT Limosilactobacillus reuteri, a probiotic microbe instrumental to human health and sustainable food production, adapts to diverse environmental shifts via dynamic gene expression. We applied the independent component analysis (ICA) to 117 RNA-seq data sets to decode its transcriptional regulatory network (TRN), identifying 35 distinct signals that modulate specific gene sets. Our findings indicate that the ICA provides a qualitative advancement and captures nuanced relationships within gene clusters that other methods may miss. This study uncovers the fundamental properties of L. reuteri’s TRN and deepens our understanding of its arginine metabolism and the co-regulation of riboflavin metabolism and fatty acid conversion. It also sheds light on conditions that regulate genes within a specific biosynthetic gene cluster and allows for the speculation of the potential role of isoprenoid biosynthesis in L. reuteri’s adaptive response to environmental changes. By integrating transcriptomics and machine learning, we provide a system-level understanding of L. reuteri’s response mechanism to environmental fluctuations, thus setting the stage for modeling the probiotic transcriptome for applications in microbial food production. IMPORTANCE We have studied Limosilactobacillus reuteri, a beneficial probiotic microbe that plays a significant role in our health and production of sustainable foods, a type of foods that are nutritionally dense and healthier and have low-carbon emissions compared to traditional foods. Similar to how humans adapt their lifestyles to different environments, this microbe adjusts its behavior by modulating the expression of genes. We applied machine learning to analyze large-scale data sets on how these genes behave across diverse conditions. From this, we identified 35 unique patterns demonstrating how L. reuteri adjusts its genes based on 50 unique environmental conditions (such as various sugars, salts, microbial cocultures, human milk, and fruit juice). This research helps us understand better how L. reuteri functions, especially in processes like breaking down certain nutrients and adapting to stressful changes. More importantly, with our findings, we become closer to using this knowledge to improve how we produce more sustainable and healthier foods with the help of microbes.

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Reviewer #2 (Comments for the Author): The manuscript presents an innovative bioinformatic platform to identify genome-wide transcriptional regulatory networks in Lm. reuteri.Analyses are based on a very large RNA-sequencing dataset that were generated with a broad range of environmental conditions.While the manuscript has improved in the revision, the interpretation of the data should be more carefully grounded by a revised discussion of metabolic pathways that have not been shown to be functional in Lm. reuteri (nitrate respiration) or are unlikely to exist (isoprenoid synthesis, synthesis of linoleic acid or SCFA other than acetate, beta oxidation).This discussion should be placed in the main manuscript, not in a "supplementary discussion" section.Specific comments.line 53 to 57.The references on CLA and riboflavin synthesis pertain to Lactiplantibacillus plantarum and Lactococcus lactis but not to Lm. reuteri.line 101.While it is appreciated that the manuscript describes a very large dataset, quantitative comparisons that allow a sound statistical analysis are generally based on triplicate independent observations.Line 290.Anaerobic respiration.Anaerobic respiration requires a nitrate / nitrite reductase and an electron transfer chain.While both genes are occasionally present in strains of Lm. reuteri, it is uncertain that both are present in the same strain and are functional.Absent at least a bioinformatic confirmation that all genes that are necessary for anaerobic respiration are present, the use of the term "anaerobic respiration" is not warranted.Nitrate reductase may alternatively serve to recycle reduced cofactors that are generated in the phosphoketolase pathway.line 308.Isoprenoid biosynthesis is lactobacilli is well documented as pigmented lactobacilli synthesize the carotenoids through isoprenoid precursors.The genes in Lm. reuteri is less than 30% homologous to known isoprenoid biosynthetic genes in lactobacilli, or to any other biochemically characterized isoprenoid biosynthetic enzyme.The prediction of the role of the gene in isoprenoid biosynthesis is merely speculation.line 371.Lm. reuteri converts fatty acids but is not known to synthesize fatty acids.line 382 / 383.Lm. reuteri is not known to produce short chain fatty acids other than acetate and certainly does not synthesize linoleic acid.line 389.Unclear.Carbohydrate metabolism in Lm. reuteri is very well characterized.Malto-oligosaccharides are utilized but starch is not as the only extracellular glycosyl hydrolases are reuteransucrases, which use sucrose to synthesize reuteran while releasing fructose, and glucanotransferases, which convert starch to a soluble polymer but not to oligosaccharides which could be used as carbon source.line 409.Beta-oxidation of fatty acids for ATP generation has not been described for Lm.reuteri.As many strains of the species do not tolerate oxygen very well, beta-oxidation is an unlikely metabolic pathway in this organism.line 482.This reviewer remains unconvinced that any strain of Lm. reuteri produces isoprenoids or carotenoids from isoprenoid precursors.LINE 585.Arginine biosynthesis is an unlikely event in Lm. reuteri.line 600.Isoprenoid biosynthesis, see above.Thank you for taking the time to provide these critical and insightful comments, which have significantly bolstered the quality of our resubmitted manuscript.We have thoroughly responded to each comment that the peer reviewers provided and used these comments to improve the quality of our manuscript for future readership.For simplified reading, the comments provided by the peer reviewers will remain black, and our responses to each remark will use blue text color.Additionally, any manuscript text that was edited to address the peer reviewer's concerns will be displayed in a red text color in our newly uploaded 'Marked-Up Manuscript.' Reviewer 2: • The manuscript presents an innovative bioinformatic platform to identify genome-wide transcriptional regulatory networks in Lm. reuteri.Analyses are based on a very large RNA-sequencing dataset that were generated with a broad range of environmental conditions.While the manuscript has improved in the revision, the interpretation of the data should be more carefully grounded by a revised discussion of metabolic pathways that have not been shown to be functional in Lm. reuteri (nitrate respiration) or are unlikely to exist (isoprenoid synthesis, synthesis of linoleic acid or SCFA other than acetate, beta oxidation).This discussion should be placed in the main manuscript, not in a "supplementary discussion" section.
○ The authors are thankful for your additional comments in this second round of revision and for continuing to improve the quality of this manuscript greatly.
We We are hopeful that these additions to our manuscript have convinced the reviewer and future readers that we are not claiming the regulation of Isoprenoid Biosynthesis but the observation of isoprenoid-regulated genes with expression that is modulated by diverse nitrogenous conditions.We have made sure to remove any mention of CLA production or biosynthesis that was missed in the previous revision and made sure only to mention the conversion or transformation from linoleic acid to CLA (Line 611).We have made sure to describe acetate and not other SCFAs as being derived from L. reuteri.We have removed any inappropriate mention of beta-oxidation.This change can be found in Line 611 and Figure 3E.Discussion about these metabolic pathways and others, which have gone into greater detail in the 'Supplementary Discussion' section, have now been added to the main text.However, we would like to retain the 'Supplementary Discussion' section not only to control the Main Text word count but also to provide the readership with a compact way to read further into our predicted 'Functional modules.'The reviewer can now find Lines 566-585 to contain an expanded discussion of 3 Functional iModulons and their associated metabolic pathways (Nitrate Reductase, Iron-Sulfur Metabolism, and Isoprenoid Biosynthesis), which are unlikely to be functional or exist in L. reuteri to be added.We have also highlighted to the reader in this section and throughout the text that these Functional iModulons require further verification to tie their prediction to functional and physiological roles in L. reuteri.• line 53 to 57.The references on CLA and riboflavin synthesis pertain to Lactiplantibacillus plantarum and Lactococcus lactis but not to Lm. reuteri.○ Thank you for providing this suggestion to update our citations to describe the bioconversion of linoleic acid to conjugated linoleic acid and riboflavin production, both by L. reuteri.Reference numbers 3 and 4 have now been updated.• line 101.While it is appreciated that the manuscript describes a very large dataset, quantitative comparisons that allow a sound statistical analysis are generally based on triplicate independent observations.○ The authors are thankful that the reviewer has decided to continue the conversation about describing our 'high-quality' sequencing datasets as being based on triplicates.Therefore, we have removed the wordage for 'high-quality' in Lines 105 (replaced with 'large') and 287 (replaced with 'large').• Line 290.Anaerobic respiration.Anaerobic respiration requires a nitrate / nitrite reductase and an electron transfer chain.While both genes are occasionally present in strains of Lm. reuteri, it is uncertain that both are present in the same strain and are functional.Absent at least a bioinformatic confirmation that all genes that are necessary for anaerobic respiration are present, the use of the term "anaerobic respiration" is not warranted.Nitrate reductase may alternatively serve to recycle reduced co-factors that are generated in the phosphoketolase pathway.
○ The authors appreciate Reviewer 2's technical knowledge in supplementing our analysis, which has greatly benefitted the quality of this manuscript throughout the peer review process.Thank you for providing additional insight into our usage of 'anaerobic respiration'.We have now modified this to 'Anaerobic Fermentation' as a functional group that contains multiple iModulons which likely underlie the regulatory processes of fermentation in L. reuteri (Lines 297-298).To comply with this modification, we have updated Figure 1A with this word usage.
Figure 4E.Lm. reuteri does not produce SCFA other than acetate.Saturated long chain fatty acids are not converted to linoleic acid.the Transcriptional Regulatory Network of Probiotic L. reuteri is Enabled by Transcriptomics and Machine Learning Manuscript ID: mSystems01257-23 JF Josephs-Spaulding, A Rajput, Y Hefner, R Szubin, A Balasubramanian, G Li, D Zielinski, LJ Jahn, MOA Sommer, PV Phaneuf, and BO Palsson

Yes, the authors completely agree that the role of the genes within the 'Isoprenoid Biosynthesis & Adaptive Response' iModulon are all 'Functional iModulons' are purely speculation. We have attempted to establish this important point throughout the manuscript. All 'Functional iModulons' i.e. iModulons that are not directly related to a known transcription factor or regulator, are speculative to describe a potential function; the value of 'Functional iModulons' lies to in the future of additional studies to unravel the potential functions that we have predicted. Meanwhile, 'Functional iModulons' and their limitations are described in our manuscript (Lines 361-363 & 559-562). To further clarify to the reviewer and readership, we have added more information in Line 23, Lines 545-550, and Lines 695-701 to inform the readers that our present prediction of isoprenoid biosynthesis in L. reuteri is purely speculative.
• line 308.Isoprenoid biosynthesis is lactobacilli is well documented as pigmented lactobacilli synthesize the carotenoids through isoprenoid precursors.The genes in Lm. reuteri is less than 30% homologous to known isoprenoid biosynthetic genes in lactobacilli, or to any other biochemically characterized isoprenoid biosynthetic enzyme.The prediction of the role of the gene in isoprenoid biosynthesis is merely speculation.○•line371.Lm.reuteri converts fatty acids but is not known to synthesize fatty acids.○The

authors thank Reviewer 2 for reiterating their concerns from the previous revisions with regard to our description of 'Fatty Acid Synthesis.' We have now ensured that the wordage 'Fatty Acid Conversion' or 'Fatty Acid Regulation' is used. These changes can be found on Lines 21, 397, 444, 605, and 607
• line 389.Unclear.Carbohydrate metabolism in Lm. reuteri is very well characterized.Malto-oligosaccharides are utilized but starch is not as the only extracellular glycosyl hydrolases are reuteransucrases, which use sucrose to synthesize reuteran while releasing fructose, and glucanotransferases, which convert starch to a soluble polymer but not to oligosaccharides which could be used as carbon source.○Thank

you for this suggestion to clarify the mechanisms underlying various carbohydrate sources and interactions. Our statement now suggests that despite L. reuteri containing specific enzymatic pathways (such as reuteransucrases and glucanotransferases), this organism likely exhibits broader carbohydrate metabolic pathways. Lines 405 -411 now reflect these changes
.• line 409.Beta-oxidation of fatty acids for ATP generation has not been described for Lm.reuteri.As many strains of the species do not tolerate oxygen very well, beta-oxidation is an unlikely metabolic pathway in this organism.○Thank

you for identifying this artifact we failed to remove in our last revision. Therefore, we have adjusted this statement to reflect our citation (Lines 441-442) better
.• line 482.This reviewer remains unconvinced that any strain of Lm. reuteri produces isoprenoids or carotenoids from isoprenoid precursors.○