Determining the Effects of a Spatially Heterogeneous Selection Pressure on Bacterial Population Structure at the Sub-millimetre Scale

A key interest of microbial ecology is to understand the role of environmental heterogeneity in shaping bacterial diversity and fitness. However, quantifying relevant selection pressures and their effects is challenging due to the number of parameters that must be considered and the multiple scales over which they act. In the current study, a model system was employed to investigate the effects of a spatially heterogeneous mercuric ion (Hg²⁺) selection pressure on a population comprising Hg-sensitive and Hg-resistant pseudomonads. The Hg-sensitive bacteria were Pseudomonas fluorescens SBW25::rfp and Hg-resistant bacteria were P. fluorescens SBW25 carrying a gfp-labelled, Hg resistance plasmid. In the absence of Hg, the plasmid confers a considerable fitness cost on the host, with µ_max for plasmid-carrying cells relative to plasmid-free cells of only 0.66. Two image analysis techniques were developed to investigate the structure that developed in biofilms about foci of Hg (cellulose fibres imbued with HgCl₂). Both techniques indicated selection for the resistant phenotype occurred only in small areas of approximately 178–353 μm (manually defined contour region analysis) or 275–350 μm (daime analysis) from foci. Hg also elicited toxic effects that reduced the growth of both Hg-sensitive and Hg-resistant bacteria up to 250 μm from foci. Selection for the Hg resistance phenotype was therefore highly localised when Hg was spatially heterogeneous. As such, for this model system, we define here the spatial scale over which selection operates. The ability to quantify changes in the strength of selection for particular phenotypes over sub-millimetre scales is useful for understanding the scale over which environmental variables affect bacterial populations.