Rcs Phosphorelay Activation in Cardiolipin-Deficient Escherichia coli Reduces Biofilm Formation

Abstract

Biofilm formation is a complex process that requires a number of transcriptional, proteomic, and physiological changes to enable bacterial survival. The lipid membrane presents a barrier to communication between the machinery within bacteria and the physical and chemical features of their extracellular environment, and yet little is known about how the membrane influences biofilm development. We found that depleting the anionic phospholipid cardiolipin reduces biofilm formation in Escherichia coli cells by as much as 50%. The absence of cardiolipin activates the regulation of colanic acid synthesis (Rcs) envelope stress response, which represses the production of flagella, disrupts initial biofilm attachment, and reduces biofilm growth. We demonstrate that a reduction in the concentration of cardiolipin impairs translocation of proteins across the inner membrane, which we hypothesize activates the Rcs pathway through the outer membrane lipoprotein RcsF. Our study demonstrates a molecular connection between the composition of membrane phospholipids and biofilm formation in E. coli and suggests that altering lipid biosynthesis may be a viable approach for altering biofilm formation and possibly other multicellular phenotypes related to bacterial adaptation and survival. IMPORTANCE There is a growing interest in the role of lipid membrane composition in the physiology and adaptation of bacteria. We demonstrate that a reduction in the anionic phospholipid cardiolipin impairs biofilm formation in Escherichia coli cells. Depleting cardiolipin reduced protein translocation across the inner membrane and activated the Rcs envelope stress response. Consequently, cardiolipin depletion produced cells lacking assembled flagella, which impacted their ability to attach to surfaces and seed the earliest stage in biofilm formation. This study provides empirical evidence for the role of anionic phospholipid homeostasis in protein translocation and its effect on biofilm development and highlights modulation of the membrane composition as a potential method of altering bacterial phenotypes related to adaptation and survival.

High-throughput mapping of the phage resistance landscape in E. coli

“…These genes have not been previously associated with phage resistance. Although the physiological role of cardiolipin is still emerging, it is known that cardiolipins play an important role in outer membrane protein translocation system and membrane biogenesis [ 121 – 123 ]. A recent study showed that decreased cardiolipin levels activate Rcs envelope stress response [ 123 ].…”

“…Although the physiological role of cardiolipin is still emerging, it is known that cardiolipins play an important role in outer membrane protein translocation system and membrane biogenesis [ 121 – 123 ]. A recent study showed that decreased cardiolipin levels activate Rcs envelope stress response [ 123 ]. Down-regulation of cardiolipin transport probably results in phage resistance via increased colanic acid biosynthesis, but further mechanistic studies are needed.…”

Cardiolipin is required in vivo for the stability of bacterial translocon and optimal membrane protein translocation and insertion

“…3), which would impair protein translocation into and across the IM. Recently, it was demonstrated that a reduction in CL levels impairs translocation across the IM of alkaline phosphatase expressed under a regulated promoter and monomeric OM OmpA protein of E. coli cells 53 grown under nutrient limited condition. CL-containing cells produce mature OmpA at a much faster rate than CL-depleted ΔclsABC1 cells 53 .…”

“…Recently, it was demonstrated that a reduction in CL levels impairs translocation across the IM of alkaline phosphatase expressed under a regulated promoter and monomeric OM OmpA protein of E. coli cells 53 grown under nutrient limited condition. CL-containing cells produce mature OmpA at a much faster rate than CL-depleted ΔclsABC1 cells 53 . The authors suggested that these bulk secretion and folding defects in turn can somehow activate the Rcs pathway via the OM lipoprotein RcsF.…”

“…bacterial secretory proteins destined beyond the IM are secreted post-translationally by the SecYEG translocon 50 , E. coli alkaline phosphatase and OmpA are widely used model proteins, which are capable for both co-and post-translational modes in vivo and in vitro 51,52 . Measurement of alkaline phosphatase enzymatic activity in intact toluene permeabilized cells is a reliable assay of translocation of preproteins across IM and enzymatic activity is used very often to quantitate the extent of translocon-assisted translocation in vivo 53 . Activity of this protein directly reflects translocation across the IM since alkaline phosphatase becomes active only after dimerization with the formation of two intramolecular disulfide bonds within each monomer in the periplasm 54 .…”