Coculturing bacteria leads to reduced phenotypic heterogeneities

Abstract

19Isogenic bacterial populations are known to exhibit phenotypic heterogeneity at the 20 single cell level. Because of difficulties in assessing the phenotypic heterogeneity of a 21 65 5 difficult to assess the heterogeneity of a single taxon within a mixed community. 66 Recently, several experimental approaches that assess the metabolic diversity of a 67 single taxon in natural communities have been developed (19, 20). However, these 68 approaches rely on FISH-probes that bind to 16S rRNA gene sequences for 69 identification of the taxon of interest. Hence, they do not allow to exclude the possibility 70 that some of the observed phenotypic differences are caused by minor genetic 71 differences between bacteria with very similar 16S rRNA genes. 72Two laser-based methods that are suitable for assessing phenotypes are flow cytometry 73 and Raman spectroscopy (21)(22)(23). Two types of light can be detected by the flow 74 cytometer, that is scattered light and fluorescence. The scattered light provides 75 information about the basic characteristics of the cells (e.g. size, shape and surface 76 properties), while the fluorescence data provides additional information about the cell 77 properties for which it has been stained (e.g. nucleic acid content, metabolic activity, 78 etc.) (24). Flow cytometry thus gives information regarding morphological as well as 79 specific physiological properties of single-cells. The Raman spectrum of a single cell 80 consists of a combination of the individual spectra of all the compounds that make up 81 this cell (e.g. proteins, nucleic acids, fatty acids, etc.). This results in a complex spectrum, 82 which can be interpreted as a chemical fingerprint of the cell (25, 26). Hence, single-cell 83 Raman spectra offer an in depth view on the biochemical composition of each 84 phenotype. 85A tool that can help to answer questions that are difficult to study directly in natural 86 communities is a synthetic ecosystem. A synthetic ecosystem consists of a selected set 87 of species under specific conditions. They are controllable and have a reduced 88 complexity in comparison to natural communities (27). Hence, they provide a way to 89 6 test ecological theories in order to better understand the rules of nature (28). A specific 90 setup for these synthetic ecosystems are co-cultures. The principle of such a system is 91 that two or more bacterial populations are cultivated together with some degree of 92 contact between them, which allows to study their interactions (29). 93An unanswered question, and the focus of this study, is whether bacteria in mixed 94 communities influence the phenotypic heterogeneity of their community partners. Here, 95 we used a synthetic community setup where two isolates were used as model 96 organisms. Four synthetic communities were created. The isolates were grown in 97 axenic cultures as a reference for non-interacting genotypes. To be able to study the 98 individual community members separately after they have been interacting via their 99 joint medi...

Quantifying synthetic bacterial community composition with flow cytometry: efficacy in mock communities and challenges in co-cultures

“…The wrongly predicted strains can be interpreted as additional phenotypes of the strains that make up the co-culture, which is in accordance with previous research. It was shown that phenotypes of bacteria change when co-cultured (52,53).…”

“…Moreover, the technique is cheap, where a sample can be analyzed for less than 1 euro. However, flow cytometry measures the phenotypic state of cells, which can lead to different fingerprints for the same bacterial strain when cultured under different conditions or when sampled in different growth phases (38,(52)(53)(54). This could prove a problem when trying to determine the composition of bacterial communities, especially when bacteria are co-cultured.…”