Heterogeneous selection in a spatially structured environment affects fitness tradeoffs of plasmid carriage in pseudomonads

Abstract

Environmental conditions under which fitness tradeoffs of plasmid carriage are balanced to facilitate plasmid persistence remain elusive. Periodic selection for plasmid-encoded traits due to the spatial and temporal variation typical in most natural environments (such as soil particles, plant leaf and root surfaces, gut linings, and the skin) may play a role. However, quantification of selection pressures and their effects is difficult at a scale relevant to the bacterium in situ. The present work describes a novel experimental system for such fine-scale quantification, with conditions designed to mimic the mosaic of spatially variable selection pressures present in natural surface environments. The effects of uniform and spatially heterogeneous mercuric chloride (HgCl(2)) on the dynamics of a model community of plasmid-carrying, mercury-resistant (Hg(r)) and plasmid-free, mercury-sensitive (Hg(s)) pseudomonads were compared. Hg resulted in an increase in the surface area occupied by, and therefore an increase in the fitness of, Hg(r) bacteria relative to Hg(s) bacteria. Uniform and heterogeneous Hg distributions were demonstrated to result in different community structures by epifluorescence microscopy, with heterogeneous Hg producing spatially variable selection landscapes. The effects of heterogeneous Hg were only apparent at scales of a few hundred micrometers, emphasizing the importance of using appropriate analysis methods to detect effects of environmental heterogeneity on community dynamics. Heterogeneous Hg resulted in negative frequency-dependent selection for Hg(r) cells, suggesting that sporadic selection may facilitate the discontinuous distribution of plasmids through host populations in complex, structured environments.

FIG. 1.

Schematic showing the apparatus used to apply cellulose fibers imbued with HgCl₂ onto a black Isopore polycarbonate membrane filter (0.2-μm pore size, 25-mm diameter; Millipore, United Kingdom), preinoculated with Hg^r and Hg^s bacteria and supported on 0.7 R2A (Oxoid, United Kingdom). A pressurized container (Invertible Air Duster; Viking, The Netherlands) was used to atomize and direct fibers toward the membrane surface.

FIG. 2.

Epifluorescence microscope image of a field of an FOV of a membrane filter showing a single cellulose fiber imbued with 12,500 μg HgCl₂ g cellulose⁻¹ surrounded by green (Hg^r) and red (Hg^s) colonies and with areas with no bacterial cover appearing as black. The membrane was inoculated with 1.13 × 10⁵ bacterial cells at a 5:4 Hg^r-to-Hg^s ratio and then sprayed with 20 mg Hg-cellulose fibers. The contrast and color balance of the image were changed with Simple PCI 6 imaging software (Compix Inc. Imaging Systems) and Adobe Photoshop CS2 (Adobe Systems Inc.). Scale bar represents 100 μm.

FIG. 3.

Proportional area of FOVs occupied by Hg^s (gray) and Hg^r (open) bacteria (a) and average number of generations undergone by Hg^s and Hg^r populations on whole membranes (b) 3 days after inoculation with approximately 10⁵ bacteria at a 1:1 Hg^r-to-Hg^s ratio. At the scale of the FOV, there is an increase in positive selection for Hg^r bacteria with the Hg concentration in both uniform (*, HgCl₂ added to agar [μM]) and heterogeneous (†, cellulose fibers imbued with HgCl₂ [μg HgCl₂ g cellulose⁻¹]) Hg treatments. Higher Hg concentrations also result in increased areas without bacterial cover (black) in heterogeneous Hg treatments only. At the scale of the whole membrane, there is an increase in positive selection for Hg^r bacteria with the Hg concentration in uniform Hg treatments but not in heterogeneous treatments. Error bars indicate the standard error of four or five replicate membranes per treatment.

FIG. 4.

The mean proportional area of FOVs occupied by Hg^s (gray) bacteria decreases and that of Hg^r (open) bacteria increases with a heterogeneous Hg treatment (9,375 μg HgCl₂ g cellulose⁻¹) compared to a no-Hg control treatment. The magnitude of this effect is greater when Hg^r bacteria are initially comparatively rare (1:1 Hg^r-to-Hg^s ratio) than when they are common (16:1 Hg^r-to-Hg^s ratio), indicating negative-frequency-dependent selection for Hg^r bacteria. The areas of FOVs without bacterial cover (black) are also shown. Error bars indicate the standard error of three replicate membranes per treatment.

FIG. 5.

Fluorescently labeled SBW25R::rfp cells (gray) exhibit more-than-threefold greater adherence to hexadecane than do wild-type SBW25R cells (gray, oblique lines), fluorescently labeled SBW25R(pQBR103::gfp) cells (open), and wild-type SBW25R(pQBR103) cells (open, oblique lines), which is indicative of higher cell surface hydrophobicity.