Socializing at the Air-Liquid Interface: a Functional Genomic Analysis on Biofilm-Related Genes during Pellicle Formation by Escherichia coli and Its Interaction with Aeromonas australiensis

To date, most attention has focused on biofilm formation on solid surfaces. By comparison, the knowledge on pellicle formation at the air-liquid interface is more limited and few studies document how bacteria decide on whether to form biofilms on solid surfaces or pellicles at the air-liquid interface to the surface-associated biofilms at the bottom. Pellicles are biofilms that form at the air-liquid interface. We demonstrated that specific strains of Escherichia coli formed pellicles in single cultures when cocultured with Carnobacterium maltaromaticum and E. coli O157:H7 but not with Aeromonas australiensis. Therefore, a combination of comparative genomic, mutational, and transcriptome analyses were applied to identify the unique genes in pellicle formation and investigate gene regulation under different growth phases. Here, we report that pellicle-forming strains do not harbor unique genes relative to non-pellicle-forming strains; however, the expression level of biofilm-related genes differed, especially for the genes encoding curli. Further, the regulatory region of curli biosynthesis is phylogenetically different among pellicle- and non-pellicle-forming strains. The disruption on modified cellulose and regulatory region of curli biosynthesis abolished pellicle formation in strains of E. coli. Besides, the addition of quorum sensing molecules (C-4-homoserine lactones [C-4-HSL]), synthesized by Aeromonas species, to pellicle formers abolished pellicle formation and implied a role of quorum sensing on pellicle formation. The deletion of autoinducer receptor sdiA in E. coli did not restore pellicle formation when cocultured with A. australiensis but modulated expression level of genes for curli and cellulose biosynthesis, resulting in a thinner layer of pellicle. Taken together, this study identified genetic determinants for pellicle formation and characterized the switching between pellicle to surface-associated biofilm in a dual-species environment, facilitating better understanding of the mechanisms for pellicle formation in E. coli and related organisms.IMPORTANCE To date, most attention has focused on biofilm formation on solid surfaces. By comparison, the knowledge on pellicle formation at the air-liquid interface is more limited and few studies document how bacteria decide on whether to form biofilms on solid surfaces or pellicles at the air-liquid interface to the surface-associated biofilms at the bottom. In this report, we characterized the regulation of biofilm-related genes during pellicle formation and document that interspecies communication via quorum sensing contributes to regulating the switch from pellicle to surface-associated biofilm. The discoveries expand the current view of regulatory cascades associated with pellicle formation.