Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

Abstract

The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiledcoil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.Osmotic pressure changes elicit transmembrane water fluxes that concentrate or dilute the cytoplasm of living cells, disrupting their structure and function. Cells respond by actively adjusting the distributions of selected solutes across the cytoplasmic membrane and water follows, restoring cellular hydration and volume (1). Bacteria can use K ϩ as an osmoregulatory solute but they prefer protein-stabilizing organic osmolytes like proline, glycine, glycine betaine, and ectoine (1, 2). These compounds are also called osmoprotectants because, when provided exogenously, they stimulate bacterial growth in high (but not low) osmotic pressure media. Well characterized, functionally redundant transporters, enzymes, and channels modulate the osmolyte composition of Gram-negative bacterium Escherichia coli (3-5). We are exploiting that system to learn how osmotic pressure is sensed, how resulting signals are transduced, and how cells respond by modulating their own structure, growth, and division.The mole fractions of the major phospholipids in the cytoplasmic membrane of E. coli are usually cited as 0.75 for phosphatidylethanolamine (PE), 3 0.20 for phosphatidylglycerol (PG), and 0.05 for ...

Salinity-Dependent Impacts of ProQ, Prc, and Spr Deficiencies on Escherichia coli Cell Structure

“…The osmolality at which ProP activity is half maximal (⌸ 1/2 /RT [ Fig. 10 legend]) is directly proportional to the anionic phospholipid content of the membrane in which ProP resides (67,68). Prc deficiency lowers the amplitude of the osmotic activation response approximately 30% and raises the osmolality required to reach half maximal activity (Fig.…”

“…Arrows denote samples to which trehalose (5 nmol) was added as a standard. dependent manner, whereas the membrane-integral ProW component of the ProU system does not (28,68,69). The effects of Prc deficiency on ProP activity occur in bacteria cultivated at moderate salinities that do not elicit spherical cell formation.…”

Induction of extracytoplasmic function sigma factors in Bacillus subtilis cells with membranes of reduced phosphatidylglycerol content

“…The anti-sigma factors might be in disorder because of a conformational change of the transmembrane region of the anti-sigma after reduction of PG, or through activation of certain membrane bound proteinases required for breakdown of the anti-sigma factors, similar to the disarray observed on a transmembrane segment of LacY in E. coli membrane lacking phosphatidylethanolamine (Dowhan et al, 2004). The activity of osmosensory transporter ProP in E. coli has been shown to be tuned with contents of PG and cardiolipin by interaction of C-terminal coiled-coil domain with these acidic phospholipids (Romantsov et al, 2008). We hope that the investigation of the new envelope stress constituted by reduction of PG content may shed light on the molecular mechanism of the induction of the ECF sigma factors.…”

Characterization and subcellular localization of a bacterial flotillin homologue

“…Several proteins are known to localize exclusively to pole regions. Examples include the pole-determining protein DivIVA (Marston et al, 1998), bacterial chemoreceptors (Alley et al, 1992), transport proteins (Romantsov et al, 2008) and many more. Other membrane proteins, such as peptidoglycan-synthesizing proteins, localize in helical patterns along the cylinder of rod-shaped cells (Daniel & Errington, 2003).…”

“…Various functions have been attributed to CL in bacterial cells. Negatively charged lipids such as PG and CL have been shown to play a role in the recruitment of membrane-integral proteins that contain a charged amphitropic helix (Matsumoto, 2001), in the activation of sensory proteins such as KdpD (Stallkamp et al, 1999) and in the localization and control of transporters involved in the osmostress response (Romantsov et al, 2008), and they are thought to be involved in the formation of nonbilayer structures that promote membrane invagination and fusion during division and sporulation events (Dowhan, 1997). Although the existence of heterogeneous lipid distribution in bacterial membranes is established, there is still controversy about lipid rafts in eukaryotic cells (Jacobson et al, 2007;Simons & Ikonen, 1997).…”