Cell-to-cell bacterial interactions promoted by drier conditions on soil surfaces Robin Tecona,1, Ali Ebrahimia,2, Hannah Kleyera, Shai Erev Levia, and Dani Ora aSoil & Terrestrial Environmental Physics, Department of Environmental Systems Science, Swiss Federal Institute of Technology (ETH) Zürich, 8092 Zürich, Switzerland Edited by Steven E. Lindow, University of California, Berkeley, CA, and approved August 13, 2018 (received for review May 15, 2018) Bacterial cell-to-cell interactions are in the core of evolutionary and 100 cells within a radius of 20 μm). For certain high cell density ecological processes in soil and other environments. Under most “hotspots,” e.g., in the rhizosphere (19), the number of neighbors conditions, natural soils are unsaturated where the fragmented can increase significantly. Although the distance to the nearest aqueous habitats and thin liquid films confine bacterial cells within neighboring cell can be relatively small on average (∼10 μmin small volumes and close proximity for prolonged periods. We report densely populated topsoils), it is highly spatially variable, and, effects of a range of hydration conditions on bacterial cell-level since soil bacterial distributions show a high degree of clustering, interactions that are marked by plasmid transfer between donor colonized microsites are often spatially isolated (19). To sum up, and recipient cells within populations of the soil bacterium Pseudo- the level of bacterial cell-to-cell interactions in soil may be far monas putida. Using hydration-controlled sand microcosms, we more limited than would appear from the high cell density values 9– 10 · −1 demonstrate that the frequency of cell-to-cell contacts under pre- commonly reported (10 10 cells g ) (20) due to the high scribed hydration increases with lowering water potential values surface area per volume of soil and the clustered spatial distri- (i.e., under drier conditions where the aqueous phase shrinks and bution of soil bacteria. In this background of limited opportu- nities for cell-to-cell interactions, additional environmental fragments). These observations were supported using a mechanistic factors regulate the frequency and duration of cell-to-cell en- individual-based model for linking macroscopic soil water potential counters in soil. Developing a better mechanistic understanding to microscopic distribution of liquid phase and explicit bacterial cell of how key factors affect encounters among bacterial cells is interactions in a simplified porous medium. Model results are in important for revealing basic principles that govern soil micro- good agreement with observations and inspire confidence in the bial community diversity, dynamics, and functioning. underlying mechanisms. The study highlights important physical In this study, we hypothesize that the probability of bacterial factors that control short-range bacterial cell interactions in soil cell-to-cell encounters and interactions on hydrated rough sur- and on surfaces, specifically, the central role of the aqueous phase faces such as found in soil is modified by soil properties and in mediating bacterial interactions and conditions that promote ge- hydration conditions. We thus seek to link macroscopic variables netic information transfer in support of soil microbial diversity. such as the soil water potential, soil pore spaces, and internal surface area to biological interactions that take place in aqueous conjugation | soil physics | vadose zone | Pseudomonas putida | HGT microhabitats. Specifically, we propose that cell-to-cell bacterial acterial cell-to-cell interactions sustain key evolutionary and Significance Becological processes in all environments, including horizon- tal gene transfer mediated by conjugative pili (1, 2), nutrient Despite a high number of microbial cells and species present in cross-feeding (e.g., in syntrophy; ref. 3), and chemical signaling small volumes of soil, detailed observations suggest that most that shapes social behaviors (4). To interact, microorganisms bacteria interact with only a few other individuals. These inter- have to remain in direct physical contact or within a range per- actions between cells are crucial to many soil processes including mitting effective molecular exchanges by diffusion, i.e., most imparting genetic traits (e.g., antibiotic resistance) and microbial MICROBIOLOGY interactions occur at the scale of individual microbes (5). Ulti- evolution, yet our understanding of the controlling environmental mately, cumulative cell-to-cell exchanges determine the overall factors remains sketchy. Based on evidence from experiments in bacterial activity in a given habitat, affecting large-scale fluxes soil microcosms and results of a mathematical model, we dem- and, hence, impacting global ecosystem processes (6). onstrate that a ubiquitous physical factor such as fragmentation Bacteria that inhabit natural porous media such as soil expe- of the aqueous phase commonly found in unsaturated soils af- rience life in a complex 3D pore network and on rough surface fects the ranges and frequency of cell-to-cell bacterial interactions. architecture, with heterogeneous nutrient resources and frag- Our findings thus help reveal some of the basic principles that mented aqueous niches that limit their distribution, dispersion, control microbial life and diversity in soil environments. and contact with neighbors, but can locally increase cell density and proximity (7–11). Evidence suggests that (i) the distribution Author contributions: R.T., A.E., H.K., S.E.L., and D.O. designed research; A.E. designed the of microorganisms in soil is highly patchy, with nonrandom mi- mathematical model; R.T., A.E., H.K., and S.E.L. performed research; R.T., A.E., H.K., and crohabitats colonized by single cells or microcolonies (12–15), S.E.L. analyzed data; and R.T., A.E., and D.O. wrote the paper. and that (ii) although soil microbial density is high in comparison The authors declare no conflict of interest. with other habitats, cells occupy no more than a few percent of This article is a PNAS Direct Submission. the pore spaces of an average soil and only a small fraction Published under the PNAS license. < ( 1%) of the available soil surfaces (9, 16, 17). This spatial Data deposition: The Modeling data and code have been deposited in the ETH Research context—together with microbial growth and motility (18)— Collection, https://www.research-collection.ethz.ch/handle/20.500.11850/284650 (DOI: 10. controls the probability for a bacterial cell to encounter another 3929/ethz-b-000284650). cell, and therefore to interact, in soil. Based on a detailed 1To whom correspondence should be addressed. Email: [email protected]. analysis of bacterial distributions in hundreds of soil thin sections 2Present address: Ralph M. Parsons Laboratory for Environmental Science and Engineer- and on statistical modeling, Raynaud and Nunan (19) have ing, Department of Civil and Environmental Engineering, Massachusetts Institute of concluded that the potential for cell-to-cell interactions in soil is Technology, Cambridge, MA 02139. relatively limited, both in terms of the number of interacting cells This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and of the number of different species to interact with. The vi- 1073/pnas.1808274115/-/DCSupplemental. cinity of a soil bacterium typically harbors few neighbors (10 to Published online September 12, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1808274115 PNAS | September 25, 2018 | vol. 115 | no. 39 | 9791–9796 Downloaded by guest on September 29, 2021 interactions could be promoted under hydration conditions that locally increase the likelihood and duration of cell encounters while limiting cell dispersal. To systematically investigate this question, we have used the exchange of a conjugative plasmid as a proxy for close encounters between bacterial cells, with the soil bacterium Pseudomonas putida (21) selected as a donor and recipient of a broad-host range plasmid isolated from soil envi- ronments (Fig. 1). Sand microcosms were used as simple and well-defined porous environments for assessing conjugation events as function of hydration conditions, as determined by a prescribed matric potential (Fig. 2 and SI Appendix, Supple- mentary Methods for details). In unsaturated soil, the matric potential results from capillary and adsorptive interactions that retain water in pores and within roughness elements (22, 23). The matric potential is often expressed as a negative pressure (relative to atmospheric pressure): where zero value marks complete water Fig. 2. Sand microcosms with controlled hydration conditions. (A) Photo- saturation, while progressively negative values correspond to drier graph shows an assembled system, with microcosms connected to a liquid conditions (22, 23). We use the simple experimental system (Figs. medium reservoir (0.1× TSB). The height of the liquid column (h) prescribes a 1 and 2) to directly study the relation between a biological cell-to- fixed suction to the sand phase via the ceramic plate, which mimics the ef- cell interaction (bacterial conjugation) and a physical parameter fects of matric potential in porous media (e.g., h = 10-cm produces a water (matric potential) relevant to soil. In addition to experiments, matric potential of ∼−1 kPa). Inset shows stereomicroscope image of the mathematical modeling provides a means for generalization and quartz sand layer. (B) Illustration of the microcosm unit,