Spatial alanine metabolism determines local growth dynamics of Escherichia coli colonies
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Curated by eLife
Evaluation Summary:
In this manuscript the authors use a combination of transcriptomics, metabolomics, and quantitative measurements of growth to characterize the temporal and spatial distribution of cells with different metabolic states within colony of biofilms of the model bacterium Escherichia coli. They show that within the biofilm cells performing different metabolic functions are distributed in different regions of the colonies, and propose a model where nutrient cross-feeding through the amino acid alanine explains the phenotypic heterogeneity observed within the biofilm. The findings reported have potential broad implications for thinking about the spatial structure of communities of all bacterial species.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Bacteria commonly live in spatially structured biofilm assemblages, which are encased by an extracellular matrix. Metabolic activity of the cells inside biofilms causes gradients in local environmental conditions, which leads to the emergence of physiologically differentiated subpopulations. Information about the properties and spatial arrangement of such metabolic subpopulations, as well as their interaction strength and interaction length scales are lacking, even for model systems like Escherichia coli colony biofilms grown on agar-solidified media. Here, we use an unbiased approach, based on temporal and spatial transcriptome and metabolome data acquired during E. coli colony biofilm growth, to study the spatial organization of metabolism. We discovered that alanine displays a unique pattern among amino acids and that alanine metabolism is spatially and temporally heterogeneous. At the anoxic base of the colony, where carbon and nitrogen sources are abundant, cells secrete alanine via the transporter AlaE. In contrast, cells utilize alanine as a carbon and nitrogen source in the oxic nutrient-deprived region at the colony mid-height, via the enzymes DadA and DadX. This spatially structured alanine cross-feeding influences cellular viability and growth in the cross-feeding-dependent region, which shapes the overall colony morphology. More generally, our results on this precisely controllable biofilm model system demonstrate a remarkable spatiotemporal complexity of metabolism in biofilms. A better characterization of the spatiotemporal metabolic heterogeneities and dependencies is essential for understanding the physiology, architecture, and function of biofilms.
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Evaluation Summary:
In this manuscript the authors use a combination of transcriptomics, metabolomics, and quantitative measurements of growth to characterize the temporal and spatial distribution of cells with different metabolic states within colony of biofilms of the model bacterium Escherichia coli. They show that within the biofilm cells performing different metabolic functions are distributed in different regions of the colonies, and propose a model where nutrient cross-feeding through the amino acid alanine explains the phenotypic heterogeneity observed within the biofilm. The findings reported have potential broad implications for thinking about the spatial structure of communities of all bacterial species.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive …
Evaluation Summary:
In this manuscript the authors use a combination of transcriptomics, metabolomics, and quantitative measurements of growth to characterize the temporal and spatial distribution of cells with different metabolic states within colony of biofilms of the model bacterium Escherichia coli. They show that within the biofilm cells performing different metabolic functions are distributed in different regions of the colonies, and propose a model where nutrient cross-feeding through the amino acid alanine explains the phenotypic heterogeneity observed within the biofilm. The findings reported have potential broad implications for thinking about the spatial structure of communities of all bacterial species.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript "Spatial alanine metabolism determines local growth dynamics of Escherichia coli colonies" by Dias-Pascual, et. al., the authors present a detailed and comprehensive spatial and temporal characterization of the molecular mechanisms involved in E. coli colony biofilm formation. With their studies the authors propose a nutrient cross-feeding model explaining the phenotypic heterogeneity observed within the biofilm. They propose that in nutrient rich regions of the biofilm where carbon and nitrogen sources are not limiting, but oxygen is limited, cells secrete alanine, this amino acid is then used by the surface (and oxygen) exposed regions of the biofilms. Mutants impaired in cross-feeding through impairment of alanine secretion and degradation, have a higher number of dead cells in the …
Reviewer #1 (Public Review):
In this manuscript "Spatial alanine metabolism determines local growth dynamics of Escherichia coli colonies" by Dias-Pascual, et. al., the authors present a detailed and comprehensive spatial and temporal characterization of the molecular mechanisms involved in E. coli colony biofilm formation. With their studies the authors propose a nutrient cross-feeding model explaining the phenotypic heterogeneity observed within the biofilm. They propose that in nutrient rich regions of the biofilm where carbon and nitrogen sources are not limiting, but oxygen is limited, cells secrete alanine, this amino acid is then used by the surface (and oxygen) exposed regions of the biofilms. Mutants impaired in cross-feeding through impairment of alanine secretion and degradation, have a higher number of dead cells in the surface exposed aerobic region of the biofilm.
The manuscript is technically very solid. The authors use a combination of temporal-spatial transcriptomics and metabolomics in addition to novel methods to measure growth and cell viability within the biofilm. These methods can be applied to study biofilms in other bacteria.
Overall, this study highlights the importance of nutrient cross-feeding within biofilm colonies, which according to the authors is likely to be important for biofilms in other systems, including multi-species biofilms, because cross-feeding interactions have already been described in multi-species bacterial interactions. Therefore, the current study, and the innovative methods applied here, will motivate novel studies to address the importance of nutrient cross-feeding interactions in other single, as well as, multi-species biofilms.
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Reviewer #2 (Public Review):
The manuscript by Diaz-Pascual et al shows that alanine metabolism varies spatially across a colony, and is related to both growth dynamics and the presence of oxygen. The authors use a combination of transcriptomics, metabolomics, and quantitative measurements of growth at the single-cell and colony levels to link these properties, all of which have potentially broad impact for thinking about the spatial structure of communities of all bacterial species.
The data supporting the links between oxygen and alanine secretion/metabolism is convincing, and the authors provide genetic evidence that links alanine usage to both carbon and nitrogen metabolism.
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Reviewer #3 (Public Review):
In the work, spatial alanine metabolism determines local growth dynamics of Escherichia coli colonies, Diaz-Pascual et all used a transcriptomic-metabolomic approach on E. coli biofilms to show that alanine production occurs in different regions of the biofilm than alanine consumption and that this alanine cross-feeding mechanism is important for biofilm growth.
The mayor strength of this work is the optimal combination of transcriptomics and metabolomics over time during bacterial growth. In my opinion, it is a technological hallmark in the study of biofilms. In my opinion, the weakness lies on certain concepts that are presented and are difficult to understand.
A number of studies show that glucose and oxygen gradients give rise to distinct metabolic phenotypes within biofilms. A glucose-rich environment …
Reviewer #3 (Public Review):
In the work, spatial alanine metabolism determines local growth dynamics of Escherichia coli colonies, Diaz-Pascual et all used a transcriptomic-metabolomic approach on E. coli biofilms to show that alanine production occurs in different regions of the biofilm than alanine consumption and that this alanine cross-feeding mechanism is important for biofilm growth.
The mayor strength of this work is the optimal combination of transcriptomics and metabolomics over time during bacterial growth. In my opinion, it is a technological hallmark in the study of biofilms. In my opinion, the weakness lies on certain concepts that are presented and are difficult to understand.
A number of studies show that glucose and oxygen gradients give rise to distinct metabolic phenotypes within biofilms. A glucose-rich environment promotes acetate-producing fermentative metabolism; a glucose-deprived environment, however, promotes acetate consumption. This is consistent with classical experiments in liquid cultures; cells grow exponentially using substrate-level phosphorylation and when the concentration of glucose decreases use other non-preferred catabolizable substrates (acetate or alanine) via TCA cycle. As E. coli cells cross-feed acetate within biofilm, a similar pattern should occur with alanine metabolism, which is the subject of this study.
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