Periodic Reporting for period 1 - MultiMEc (A multilevel integrative approach to microbial ecology: from molecular networks to cellular interactions in a spatially structured community)
Período documentado: 2021-08-01 hasta 2023-07-31
Microbial communities profoundly influence global biogeochemical cycles and human life. Understanding their wiring is crucial to manage, rationally manipulate, or de novo assemble communities for environmental, industrial or medical applications. However, studying the complex web of microbial interactions and how they are affected by the spatial structure of the community is experimentally challenging. A crucial yet poorly understood aspect of microbial interactions is the spatial dimension. For example, in a biofilm or the intestinal mucosae in the human gut, bacteria find themselves in a highly structured environment where each cell is exposed to a unique set of environmental factors depending on its position in the community and its immediate neighbours. To better understand microbial communities, we need to know how individual cells interact with each other and how molecular and cellular processes in individuals scale up to determine the structure and activity of the entire community. The goal of the proposed research was to study microbial interactions at multiple levels, from the molecular processes involved, to the effects of these interactions on survival and growth, to how they are affected by environmental conditions and the spatial arrangement of cells.
To achieve this, we worked with a synthetic community consisting of two members of the human gastrointestinal microbiota, the commensal gut microbe Escherichia coli and the lactic acid bacterium Lactobacillus plantarum. L. plantarum is widely used in food fermentation and has been reported to have a number of health benefits. As is typical for lactic acid bacteria, L. plantarum has multiple amino acid and vitamin auxotrophies; however, these may be compensated for by co-culturing it with E. coli which produces the required compounds. Recent mathematical models suggest that the two species engage in diverse metabolic interactions in the gut and that these interactions drastically differ as a function of oxygen availability.
The project established the synthetic community of the two gut microbial strains E. coli and L. plantarum as a model system for future studies by us and others. By profiling different growth conditions and performing follow-up experiments in a spatially structured setting, we found conditions in which the two strains interact in a mutualistic way, i.e. both are benefiting of the presence of the other strain. We also made an interesting discovery where we found that L. plantarum can assume a “zombie” state in which it is itself unable to grow but metabolically active enough to support E. coli’s growth over extended periods of time. Such a phenomenon has, to the best of our knowledge, not yet been described before for any bacterial species and our research group is continuing the research on this project.
To achieve this, we worked with a synthetic community consisting of two members of the human gastrointestinal microbiota, the commensal gut microbe Escherichia coli and the lactic acid bacterium Lactobacillus plantarum. L. plantarum is widely used in food fermentation and has been reported to have a number of health benefits. As is typical for lactic acid bacteria, L. plantarum has multiple amino acid and vitamin auxotrophies; however, these may be compensated for by co-culturing it with E. coli which produces the required compounds. Recent mathematical models suggest that the two species engage in diverse metabolic interactions in the gut and that these interactions drastically differ as a function of oxygen availability.
The project established the synthetic community of the two gut microbial strains E. coli and L. plantarum as a model system for future studies by us and others. By profiling different growth conditions and performing follow-up experiments in a spatially structured setting, we found conditions in which the two strains interact in a mutualistic way, i.e. both are benefiting of the presence of the other strain. We also made an interesting discovery where we found that L. plantarum can assume a “zombie” state in which it is itself unable to grow but metabolically active enough to support E. coli’s growth over extended periods of time. Such a phenomenon has, to the best of our knowledge, not yet been described before for any bacterial species and our research group is continuing the research on this project.
When we started our research, the synthetic two-strain community of E. coli and L. plantarum had not yet been established as a model system and we first worked towards developing protocols and identifying optimal conditions to culture the strains individually and combined in the lab under different growth conditions that are relevant for the proposed work. We then established the plasmids and protocols for the genetic engineering of L. plantarum. These were important, on the one hand, to generate a fluorescent L. plantarum strain which we needed for high-throughput growth profiling and, on the other hand, for the planned genetic perturbations. After extensive optimization, we identified conditions in which each strain individually cannot grow but when grown together they can support each other’s growth in well-mixed cultures; the media used is minimal and contains sucrose as a sole carbon source, which is inaccessible to E. coli. We then established a robust workflow for cultivation of the strains in microfluidic chips that allowed us to observe their growth by time-lapse imaging and obtain insights into the spatial aspects of their interaction. We found, as in the well-mixed cultures, that L. plantarum can support growth of E. coli and thanks to our experimental setup, we were able to quantify the distance over which the cross-feeding occurs, which was less than 10 µm. Interestingly, L. plantarum itself did not grow itself but apparently remained metabolically active enough to support E. coli’s growth over at least 48 hours. Due to a career opportunity, the fellowship was terminated early but our research group continues to work on this system, and we are planning to follow up with the proposed plans to use proteomics and metabolomics approaches together with genetic perturbations to dissect the mechanisms involved in the interaction between E. coli and L. plantarum in the presence and absence of oxygen.
As part of the project, we invested significantly into developing a software tool for the automated analysis of time-lapse microscopy videos that will be useful for a wider community of microbiologists. For this, we have been working closely with two professional software developers from ETH Zurich’s Scientific IT Services. So far, five research groups in Switzerland have been using this software and we expect this number to expand and include other European and international groups soon, too.
No website has been developed for the project.
As part of the project, we invested significantly into developing a software tool for the automated analysis of time-lapse microscopy videos that will be useful for a wider community of microbiologists. For this, we have been working closely with two professional software developers from ETH Zurich’s Scientific IT Services. So far, five research groups in Switzerland have been using this software and we expect this number to expand and include other European and international groups soon, too.
No website has been developed for the project.
Our research aimed at understanding the connections between genetic networks, molecular processes, and the behavior of individual bacterial cells in space as they interact in a mixed-species community. The experimental system we developed, and the findings obtained with it, e.g. the existence of a state in which bacterial community members cannot grow but still contribute to the growth of others, contribute insights towards understanding fundamental principles governing microbial interactions. Furthermore, our research suggests how the probiotic lactic acid bacterium L. plantarum may interact with other gut residents, which, in turn, may inform the design of more effective treatments for diseases thought to be related to dysbiosis of the gut microbiota.
This fellowship had a major impact on my career. Upon returning from the United States where I stayed as a postdoctoral scholar for several years, it allowed me to re-integrate into the European research landscape and establish a unique and competitive scientific profile. I now started a tenure-track group leader position at Eawag, the Swiss Institute for Aquatic Science and Technology and ETH Zurich, and therefore terminated the fellowship early.
This fellowship had a major impact on my career. Upon returning from the United States where I stayed as a postdoctoral scholar for several years, it allowed me to re-integrate into the European research landscape and establish a unique and competitive scientific profile. I now started a tenure-track group leader position at Eawag, the Swiss Institute for Aquatic Science and Technology and ETH Zurich, and therefore terminated the fellowship early.