Defining the role of intracellular signaling in interspecies interactions

Microbes form communities that govern various fundamental processes on planet Earth. Soil bacteria have numerous roles in ecological processes essential for the health and survival of plants. They provide plants with protection from pathogenic bacteria and the nutrients essential for their well-being. In addition, the interactions between soil microbes and plants control nutrient cycling and greenhouse gas emissions. In turn, these micro-organisms indirectly affect communities of animals and the surrounding ecosystem. There is knowledge surrounding the complexity of these microbial communities, and more recently, discoveries of how these microbial connections can influence community structure. While mechanisms such as quorum sensing and cross-feeding are known to contribute to interactions among bacteria, there is still a wide knowledge gap surrounding the mechanisms that facilitate interactions and how these organisms integrate their perception and contact of another species into their behavior. An interaction-induced trait known as interspecies social spreading was previously discovered between two different bacteria, Pedobacter sp. V48 and Pseudomonas fluorescens Pf0-1. We studied this pairwise interaction to gain an expanded understanding of interaction-induced traits and the mechanisms that trigger them. The c-di-GMP signaling network is predicted to be involved in interspecies social spreading (ISS). Mutants of Pseudomonas fluorescens Pf0-1 which each lack one of the c-di-GMP genes were tested for defects in ISS, and in phenotypes that may contribute to ISS (swimming motility and biofilm formation). Two genes within this signaling (Pfl01_4307 and Pfl01_5111) were shown to be important for the ISS phenotype. When co-cultured with Pedobacter sp. V48 ISS did not occur. Both mutants were defective in biofilm development, and one was incapable of swimming motility, indicating a link between these traits and ISS. To further assess the role of each gene, induction at two different levels was performed using complementation experiments in which expression of the Pseudomonas genes was controlled by a rhamnose-inducible promoter. While expression of Pfl01_5111 restored ISS in the mutant, expression of did not restore ISS at each level of induction. Interestingly, expression of Pf0-1_4307 in the wild-type P. fluorescens led to a reduction of ISS. Preliminary data suggests that Pedobacter sp. V48 may influence expression of c-di-GMP gene expression in P. fluorescens during ISS. Using a GFP reporter plasmid, the effect of Pedobacter sp. V48 on intracellular c-di-GMP in P. fluorescens and mutant strains was measured in both structured and unstructured communities. In a structured community, the presence of Pedobacter sp.V48 caused a significant increase in c-di-GMP signaling molecule solely within Pf0-1, while an unstructured community resulted in elevated levels in both Pf0-1 as well as mutant strains. The results of this study suggest that Pedobacter sp. V48 directly impacts c-di-GMP levels during ISS interaction, and that the role of specific genes within the c-di-GMP system is nuanced. We speculate that phenotypes resulting from these mutations are a result of community structure and a myriad of c-di-GMP signaling mechanisms which are discussed in this thesis.