Heterogeneity of iron bioavailability on plants assessed with a whole-cell GFP-based bacterial biosensor

Abstract

Ferric iron is an essential element for microbial growth but its water solubility in aerobic environments is considered to be low. Thus it is a limiting resource for which microbes must compete in natural habitats. Since competition for iron occurs at the level of individual cells, knowledge of the variability in iron bioavailability to such individuals is required to assess the nature of the competition in these habitats. Ferric iron availability to cells of Pseudomonas syringae was assessed by quantifying the fluorescence intensity of single cells harbouring a plasmid-borne transcriptional fusion of an iron-regulated promoter from a locus encoding a membrane receptor for a pyoverdine siderophore with a reporter gene encoding green fluorescent protein (GFP) following fluorescence microscopy. Cells of this iron biosensor exhibited irondependent GFP fluorescence that was inversely proportional to the amount of iron added to the media, and which differed by over 20-fold in iron-replete compared to iron-deplete culture media. Cells cultured in a medium of a given iron content exhibited a very narrow range of fluorescence intensities. In contrast, the fluorescence intensity of cells of the biosensor strain recovered from the rhizosphere or phylloplane of inoculated bean plants varied greatly. The distribution of fluorescence intensities was strongly right-hand skewed, with about 10 % of the cells exhibiting substantially higher GFP fluorescence than that of the median cell. Cells of a positive control strain, harbouring a fusion of the constitutive nptII promoter with the gfp reporter gene, exhibited uniform GFP fluorescence both in culture media and on plants. These results indicate that there is substantial heterogeneity of iron biovailability to cells of P. syringae on plants, with only a small subset of cells experiencing low iron availability. Such heterogeneity places constraints on models of interactions of bacteria in natural habitats that are based on competition for limited iron.

Linking environmental heterogeneity and reproductive success at single-cell resolution

“…The heterogeneity that we observed in the ability of individual immigrants to produce offspring on the leaf surface is a novel observation. It corroborates findings of others who have documented leaf-based heterogeneity in environmental stimuli (Joyner and Lindow, 2000;Leveau and Lindow, 2001a;Axtell and Beattie, 2002;Gunasekera and Sundin, 2006), each of which is likely to affect reproduction. Together, these data support the hypothesis that local conditions are the key determinants of the abundance and dynamics of microbes on plant leaf surfaces (Woody et al, 2007).…”

“…Temporal and spatial variation in single-cell green fluorescence indicated substantial heterogeneity in the availability of fructose to individual leaf colonizers (Leveau and Lindow, 2001a). Such heterogeneity has also been reported for other nutrients or stimuli that leaf bacteria are exposed to, including iron (Joyner and Lindow, 2000), water (Axtell and Beattie, 2002), UV light (Gunasekera and Sundin, 2006) and phenolic compounds (Sandhu et al, 2007).…”

The histidine utilization (hut) genes of Pseudomonas fluorescens SBW25 are active on plant surfaces, but are not required for competitive colonization of sugar beet seedlings

“…cloacae JL1157 and P. fluorescens A506 in the plant environment (Casavant et al, 2002(Casavant et al, , 2003. They also complement green fluorescent protein (GFP)-based work that has demonstrated heterogeneity in the bioavailability of sugar (Leveau & Lindow, 2001) and iron (Joyner & Lindow, 2000), as well as indoleacetic acid metabolism, in epiphytic bacteria (Brandl et al, 2001).…”

Quorum size of Pseudomonas syringae is small and dictated by water availability on the leaf surface

“…Plasmid pIRed was introduced into Pss by electroporation as in other studies (17). A second reporter strain was constructed by electroporation of the pBQ9RED plasmid into Pss B728a (pVITIR), which contains an iron starvation inducible P pvd:gfp gene fusion (44).…”