Investigating the Role of Carotenoids in Membrane Organization of <i>Pantoea</i> sp. YR343

Bacterial cell membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties to the membrane and allows it to respond to environmental conditions. Pantoea sp. YR343 is a plant-associated gram-negative gamma-proteobacteria characterized by the presence of carotenoids in the membrane. Pantoea sp. YR343 mutants lacking phytoene synthase (encoded by crtB), which catalyzes the first step in carotenoid biosynthesis, failed to produce carotenoids and displayed enhanced sensitivity to reactive oxygen species. The crtB mutant also displayed unexpected defects in biofilm formation, secretion of indole-3-acetic acid, and root colonization compared to wildtype cells. We hypothesized that the phenotypic defects observed in the crtB mutant were due to the changes in both lipid and protein composition which modulate the biophysical properties of the membrane and influence signaling and transport.Mass spectrometry analysis of the lipid head and tails revealed a significant abundance of phosphotidylethanolamine and unsaturated fatty acids in the crtB mutant cells. Using both fluorescence anisotropy (FA) and atomic force microscopy on whole cells, we showed that the crtB mutant membranes were less fluid when compared to wildtype membranes. We further characterized the membranes using spheroplasts and natural extract vesicles, which showed that presence of the outer wall does not impact membrane fluidity, but the presence of membrane proteins and carotenoids do have a strong impact. Proteomic profiling of wildtype and crtB mutant membranes revealed a significant loss of several membrane protein classes in the crtB mutant. Among the different classes of proteins, signaling and transport, outer membrane biogenesis and cell motility protein classes were the most affected in the mutant. The loss of specific protein classes may explain, at least in part, the phenotypic defects associated with the crtB mutant.Our study highlights the importance of carotenoids beyond its anti-oxidant potential. Loss of bacterial carotenoids changes membrane organization and dynamics by affecting the composition and distribution of both lipids and membrane proteins. Importantly, carotenoids regulate membrane fluidity, which is an important parameter for bacterial adaptation and survival.