Interspecies Social Spreading: Interaction between Two Sessile Soil Bacteria Leads to Emergence of Surface Motility

Abstract

Bacteria often live in complex communities in which they interact with other organisms. Consideration of the social environment of bacteria can reveal emergent traits and behaviors that would be overlooked by studying bacteria in isolation. Here we characterize a social trait which emerges upon interaction between the distantly related soil bacteria Pseudomonas fluorescens Pf0-1 and Pedobacter sp. strain V48. On hard agar, which is not permissive for motility of the monoculture of either species, coculture reveals an emergent phenotype that we term "interspecies social spreading," where the mixed colony spreads across the hard surface. We show that initiation of social spreading requires close association between the two species of bacteria. Both species remain associated throughout the spreading colony, with reproducible and nonhomogenous patterns of distribution. The nutritional environment influences social spreading: no social behavior is observed under high-nutrient conditions, but low-nutrient conditions are insufficient to promote social spreading without high salt concentrations. This simple two-species consortium is a tractable model system that will facilitate mechanistic investigations of interspecies interactions and provide insight into emergent properties of interacting species. These studies will contribute to the broader knowledge of how bacterial interactions influence the functions of communities they inhabit.IMPORTANCE The wealth of studies on microbial communities has revealed the complexity and dynamics of the composition of communities in many ecological settings. Fewer studies probe the functional interactions of the community members. Function of the community as a whole may not be fully revealed by characterizing the individuals. In our two-species model community, we find an emergent trait resulting from the interaction of the soil bacteria Pseudomonas fluorescens Pf0-1 and Pedobacter sp. V48. Observation of emergent traits suggests there may be many functions of a community that are not predicted based on a priori knowledge of the community members. These types of studies will provide a more holistic understanding of microbial communities, allowing us to connect information about community composition with behaviors determined by interspecific interactions. These studies increase our ability to understand communities, such as the soil microbiome, plant-root microbiome, and human gut microbiome, with the final goal of being able to manipulate and rationally improve these communities.

Keywords: Pseudomonas fluorescens; interspecies interaction; motility; sociomicrobiology.

FIG 1

Mixed colony of P. fluorescens Pf0-1 and Pedobacter sp. V48 spreads across a hard surface (2% agar), a behavior not observed in the monoculture of either species. (a) Diameter of colonies at 24-h intervals for three independent experiments. (b) Phenotypes of mono- and cocultures at 24-h intervals, starting at 24 h. Contrast and brightness levels were adjusted for optimal viewing.

FIG 2

Mixed colony of P. fluorescens Pf0-1 and Pedobacter sp. V48 at different magnifications. (a) Image of the whole coculture colony created by stitching an ×8 magnification mosaic. (b) ×16 magnification of the leading edge showing the patterns of “petals” (1) in between “veins” (2) visible near the edge of the colony. (c) ×112 magnification shows a terraced appearance of the leading edge. Colony imaged 144 h after inoculation. Black boxes indicate area enlarged in the adjacent panel. Scale bars are noted at the bottom of each image. Contrast and brightness levels were adjusted for optimal viewing.

FIG 3

Mixed colony of fluorescently tagged P. fluorescens Pf0-1 (Pf0-ecfp) and Pedobacter sp. V48 (V48-dsRed). (a) Coculture colony viewed with white light. (b) Coculture imaged using DsRed filter (filter set 43 HE), pseudocolored in orange, showing V48-dsRed distribution throughout the colony. (c) Coculture imaged using CFP filter (filter set 47 HE), pseudocolored in turquoise, showing Pf0-ecfp distribution throughout the colony. (d) Merged images of DsRed and CFP filters. Numbers in panels b and c indicate six zones of distinct patterns: 1, point of inoculation; 2, coffee ring; 3, starburst; 4, P. fluorescens ring; 5, petals; 6, veins. Colonies imaged at ×7 magnification; scale bar represents 1 mm. Colony imaged 144 h after inoculation.

FIG 4

Social spreading emerges after contact between colonies of P. fluorescens Pf0-1 and Pedobacter sp. V48, left and right in each panel, respectively. Colonies come into contact 24 h after inoculation; the motile front becomes visible 48 h after contact and spreads outward and around the P. fluorescens colony before surrounding the Pedobacter colony. Black spots indicate sampling locations. Pictures taken every 24 h. Scale bar represents 10 mm.

FIG 5

A semipermeable barrier prevents development of the interspecies social spreading phenotype. (a) Pedobacter sp. V48 monoculture; (b) P. fluorescens Pf0-1 monoculture; (c) a mixed colony; (d) P. fluorescens and Pedobacter separated by a mixed-ester cellulose membrane. Pictures taken 144 h after inoculation. Colonies were grown on a 100-mm petri dish.

FIG 6

Low-nutrient and high-salt conditions are required for interspecies social spreading. (a) Mixed colony on full-strength TSB does not show social spreading. (b) Mixed colony on TSB-NK (10% tryptic soy supplemented with both NaCl and KH₂PO₄) shows social spreading. (c) Mixed colony on 10% strength TSB shows impaired social spreading. (d) Mixed colony on TSB-N (supplemented with NaCl) exhibits the interspecies social spreading phenotype. For all panels, Pedobacter sp. V48 monoculture is on the top left of the plate, P. fluorescens Pf0-1 is on the top right of the plate, and the mixed colony is in the center. Pictures were taken 144 h after inoculation. Colonies were grown on a 100-mm petri dish.

FIG 7

Effect of nutrient levels on the interaction between P. fluorescens Pf0-1 and Pedobacter sp. V48, looking at 3 core components of TSB: tryptone (20 g/liter), d-glucose (2.5 g/liter), and NaCl (5 g/liter) for “high” concentrations. Components were reduced to 1/10 for “low” concentrations. For all panels, Pedobacter monoculture is on the top left of the plate, P. fluorescens is on the top right of the plate, and the mixed colony is in the center. Pictures were taken 144 h after inoculation. Colonies were grown on 100-mm petri dishes.