Exploring the relationship between lipoprotein mislocalization and activation of the Rcs signal transduction system in Escherichia coli

Abstract

The Rcs phosphorelay signal transduction system controls genes for capsule production and many other envelope-related functions and is implicated in biofilm formation. We investigated the activation of the Rcs system in a pgsA null mutant of Escherichia coli, which completely lacks the major acidic phospholipids phosphatidylglycerol and cardiolipin. We found that the Rcs activation, and consequent thermosensitivity, were suppressed by overexpression of the lgt gene, encoding diacylglyceryltransferase, which catalyses the modification of prolipoproteins that is the first step in the maturation and localization process of lipoproteins, and is a prerequisite for the later steps. The outer-membrane lipoprotein RcsF is an essential component of Rcs signalling. This lipoprotein was poorly localized to the outer membrane in the pgsA null mutant, probably because of the absence of phosphatidylglycerol, the major donor of diacylglycerol in the Lgt reaction. Even in a pgsA + background, the Rcs system was activated when RcsF was mislocalized to the inner membrane by alteration of the residues at positions 2 and 3 of its mature form to inner-membrane retention signals, or when it was mislocalized to the periplasm by fusing the mature form to maltose-binding protein. These results suggest that RcsF functions as a ligand for RcsC in activating Rcs signalling. Mislocalized versions of RcsF still responded to mutations pgsA, mdoH and tolB, further activating the Rcs system, although the rfaP mutation barely caused activation. It seems that RcsF must be localized in the outer membrane to respond effectively to stimuli from outside the cell.

Importance of the proline-rich region for the regulatory function of RcsF, an outer membrane lipoprotein component of the Escherichia coli Rcs signal transduction system

“…When the lipoprotein sorting signal at position 2 of the mature form is changed from a Ser residue to Asp (S2D), the Rcs system is activated as well. A stronger inner membrane retention signal, Asp at position 2 and Gln at position 3 (S2DM3Q) (Tokuda & Matsuyama, 2004), which leads RcsF to localize almost completely to the inner membrane, results in even higher activation (Shiba et al, 2012). Mislocalization of RcsF probably makes it more accessible to RcsC in the inner membrane.…”

“…In the pgsA null mutant the precursor form of RcsF is accumulated in the inner membrane and the Rcs system is activated (Shiba et al, 2012). Accumulation in the inner membrane is, however, not the sole cause of Rcs activation in the pgsA null mutant: RcsFS2DM3Q, which localizes completely to the inner membrane, shows a higher Rcs activation in the pgsA null mutant cells than in the pgsA + cells (Shiba et al, 2012). The pgsA mutation seems to impose another envelope stress in addition to the mislocalization of RcsF.…”

“…Mislocalization of RcsF to the periplasm, which can be achieved by fusing it to periplasmic maltose-binding protein (MBP), leads to activation of the Rcs system (Shiba et al, 2012). When the lipoprotein sorting signal at position 2 of the mature form is changed from a Ser residue to Asp (S2D), the Rcs system is activated as well.…”

“…The mutant accumulates small amounts of phosphatidic acid and cytidine diphosphate diacylglycerol (Kikuchi et al, 2000), which can serve as diacylglycerol donors in the prolipoprotein modification reaction, but the modification efficiency is very low (Sankaran & Wu, 1994;Suzuki et al, 2002). In the pgsA null mutant the precursor form of RcsF is accumulated in the inner membrane and the Rcs system is activated (Shiba et al, 2012). Accumulation in the inner membrane is, however, not the sole cause of Rcs activation in the pgsA null mutant: RcsFS2DM3Q, which localizes completely to the inner membrane, shows a higher Rcs activation in the pgsA null mutant cells than in the pgsA + cells (Shiba et al, 2012).…”

“…Mislocalization of RcsF probably makes it more accessible to RcsC in the inner membrane. It is likely that RcsF functions as a ligand for RcsC in activating Rcs signalling (Shiba et al, 2012 prerequisite for the later processing (Tokuda & Matsuyama, 2004). The predominant donor of diacylglycerol is phosphatidylglycerol, a major acidic phospholipid in E. coli (Sankaran & Wu, 1994).…”