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

University of California, Department of Plant and Microbial Biology, 111 Koshland Hall, Berkeley, CA 94720-3102, USA 1 Author for correspondence: Steven E. Lindow. Tel: +1 510 642 4174. Fax: +1 510 642 4995. e-mail: icelab{at}socrates.berkeley.edu 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 iron-dependent 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 npt II 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. Keywords: FISH, Pseudomonas syringae , siderophore, ferric iron Abbreviations: CCD, charge-coupled device; FISH, fluorescence in situ hybridization; GFP, green fluorescent protein; AMRA, tetramethylrhodamine-5-isothiocyanate