Linking environmental heterogeneity and reproductive success at single-cell resolution

Abstract

Individual-based microbial ecology (IBME) is a developing field of study in need of experimental tools to quantify the individual experience and performance of microorganisms in their natural habitats. We describe here the conception and application of a single-cell bioreporter approach with broad utility in IBME. It is based on the dilution of stable green fluorescent protein (GFP) in dividing bacteria. In the absence of de novo synthesis, GFP fluorescence of a daughter cell approximates half of that of its mother, from which follows that the fluorescence of a progeny cell is a quantitative measure for the reproductive success of its ancestor. To test this concept, we exposed GFP-filled bacteria to different degrees of environmental heterogeneity and assessed how this affected individual cells by the analysis of GFP content in their progeny. Reporter bacteria growing in rich medium in a shaking flask showed no variation in reproductive success, confirming that life in a broth is experienced much the same from one bacterium to the next. In contrast, when reporter bacteria were released onto plant leaf surfaces, representing a microscopically heterogeneous environment, clear intrapopulation differences in reproductive success were observed. Such variation suggests that individual cells in the founding population experienced different growthpermitting conditions, resulting in unequal contributions of individual bacteria to future offspring and population sizes. Being able to assess population changes bottom-up rather than top-down, the bioreporter offers opportunities to quantify single-cell competitive and facilitative interactions, assess the role of chance events in individual survivorship and reveal causes that underlie individual-based environmental heterogeneity.

Quantification of lateral heterogeneity in carbohydrate permeability of isolated plant leaf cuticles

“…Trichomes have also been identified as sites where sugars are commonly and abundantly available to leaf-colonizing bacteria (Leveau and Lindow, 2001). Heterogeneity in the availability of nutrients on the leaf surface is consistent with the recent observation that bacterial immigrants to the bean phyllosphere experience very different fates (Remus-Emsermann and Leveau, 2010): while most cells divided a few times upon arrival, a few divided many more times. The latter was explained by assuming that those cells landed in sites of high nutrients access.…”

“…The bioreporter was based on Erwinia herbicola 299 ( Eh 299), a natural colonizer of the phyllosphere (Brandl and Lindow, 1996) which has been used in many phyllosphere studies (Brandl et al, 2001; Leveau and Lindow, 2001; Krimm, 2005; Remus-Emsermann and Leveau, 2010; van der Wal and Leveau, 2011). Eh 299 is equipped with a construct allowing expression of the green-fluorescent protein (GFP) in response to fructose exposure (Leveau and Lindow, 2001).…”

A functional perspective on phenotypic heterogeneity in microorganisms

“…One such approach is to encapsulate single cells in gel microdroplets so that these cells can grow in clonal microcolonies while their chemical coupling to their microenvironment is preserved 112 . Another approach can be applied for studies on host interactions: experimental host systems allow the study of microorganisms in their natural environment -the hostunder well-controlled laboratory conditions, and such experiments have shed light on the role of phenotypic heterogeneity in these host-microorganism interactions 41,77,113 . 54 .…”

Microbiology of the phyllosphere: a playground for testing ecological concepts

“…Visualization techniques such as confocal and epifluorescence microscopy and tools based on fluorescent protein markers improve the experimental options even further. The use of green fluorescent protein (GFP) has truly revolutionized our ability to monitor the whereabouts, behaviours and interactions of individual bacterial cells colonizing the leaf surface (Leveau and Lindow 2001; Remus-Emsermann and Leveau 2010). These and other molecular tools make microbal communities of the phyllosphere more tractable for experimental tests of concepts ranging from landscape ecology and macroecology to biogeography.…”