Functional microdomains in bacterial membranes

Abstract

The membranes of eukaryotic cells harbor microdomains known as lipid rafts that contain a variety of signaling and transport proteins. Here we show that bacterial membranes contain microdomains functionally similar to those of eukaryotic cells. These membrane microdomains from diverse bacteria harbor homologs of Flotillin-1, a eukaryotic protein found exclusively in lipid rafts, along with proteins involved in signaling and transport. Inhibition of lipid raft formation through the action of zaragozic acid--a known inhibitor of squalene synthases--impaired biofilm formation and protein secretion but not cell viability. The orchestration of physiological processes in microdomains may be a more widespread feature of membranes than previously appreciated.

Figure 1.

yisP affects the pathway to biofilm formation. (A) Schematic representation of the signaling pathway leading to biofilm formation in B. subtilis. The pathway is triggered by activation of the master regulator Spo0A via phosphorylation by KinC. Dashed lines represent indirect activation. (B) Putative metabolic pathway to the formation of distinct polysioprenoids in B. subtilis. Enzymes discussed in the text are written in red, next to the reaction they catalyze. Dashed lines represent unknown steps. (C) Pellicle formation assay to test biofilm formation in different strains of B. subtilis. Positive and negative controls are represented by the wild-type strain (WT) and the matrix-deficient mutant (Δeps ΔyqxM) (Branda et al. 2004, 2006).

Figure 2.

YisP has squalene synthase activity and is involved in the production of a carotenoid. (A) Enzymatic activity of purified YisP from B. subtilis under different conditions. FPP at 3.7 μM was used as substrate under the optimal conditions specified in Supplemental Figure S2. Control reaction was performed with no enzyme added. (B) The ΔyisP mutant does not produce a dark-orange pigment associated with the cells, in comparison with the wild-type strain.

Figure 3.

Membrane fractionation and detection of KinC. (A) Membrane fractionation according to differential sensitivity to detergent solubilization. The membrane fractions sensitive and resistant to detergent solubilization are named DSM and DRM, respectively. Membrane proteins associated with each fraction are shown in an SDS-PAGE. DRM-associated proteins decreased in the ΔyisP mutant and in the wild-type strain treated with zaragozic acid (+Z lane). (B) Immunoblot analysis detecting KinC in each fraction. Numbers represent molecular weight standards in kilodaltons.

Figure 4.

Flotillin and KinC localization in B. subtilis. (A) Cellular localization of FloT-YFP (falsecolored red). (B) Colocalization of FloT and KinC in a double-labeled strain expressing the translational fusions FloT-YFP (false-colored red) and KinC-CFP (false colored green). Regions where the two signals overlapped appear yellow in the merge panel. (C) Eight-hour time course following localization of the translational fusion FloT-YFP after the addition of zaragozic acid to the sample. Bars: A,B, 1 μm; C, 2 μm.

Figure 5.

Colocalization of FloT and YqfA, the two SPFH domain proteins in B. subtilis. Deletion of floT and yqfA compromises KinC-dependent biofilm formation. (A) Colocalization of FloT and YqfA in a double-labeled strain expressing the translational fusions FloT-Yfp (false-colored in red) and YqfA-Cfp (false-colored in green). Regions where the two signals overlapped appear yellow in the merge panel. Bar, 3 μm (B) Deletion of floT and yqfA compromises KinC-dependent biofilm formation. The ΔfloT ΔyqfA double mutant was tested for its ability to form pellicles in response to the signaling molecule surfactin, when cultured in LB medium (Lopez et al. 2009). This behavior is dependent on the histidine kinase KinC, since the kinC-deficient mutant does not make pellicles when surfactin is added. The first and second rows of pictures show pellicles when surfactin was added to the wild-type strain, and the absence of pellicle formation in the kinC-deficient background, respectively. (Third row) A weak induction of pellicle formation was observed in the double mutant ΔfloT ΔyqfA when surfactin was added. (Fourth row) Overexpression of KinC in the double mutant ΔfloT ΔyqfA restored the ability to respond to surfactin by forming pellicles in the assay. (Fifth row) Introducing the allele sad67 in the double mutant ΔfloT ΔyqfA restored the ability to form pellicles.

Figure 6.

Lipid rafts in S. aureus. (A) Cellular localization of the translational fusion FloT-YFP in S. aureus (SA1402-YFP). The signal emitted is shown in red. Wild type untreated and treated with zaragozic acid are compared. Bar, 1μm. (B) Inhibition of protease secretion in S. aureus using zaragozic acid. Secretion of proteases in casein-containing medium produces a clear halo due to the degradation of casein. The colony treated with zaragozic acid also shows inhibited production of the pigmented carotenoid staphyloxanthin. Bar, 1 mm.

Figure 7.

Inhibition of biofilm formation by sterol-lowering drugs. (A) Schematic representation of the two distinct biochemical pathways to produce squalene in B. subtilis and S. aureus. Zaragozic acid acts as a competitive inhibitor in both routes, since it acts downstream from the formation of FPP. Statins such as mevastin and lovastatin inhibit the enzyme HMG-CoA reductase, and thus the route to produce squalene in S. aureus. Clomazone inhibits the enzyme 1-deoxy-D-xylulose 5-phosphate synthase, and thus the route to produce squalene in B. subtilis. (B) Addition of different concentrations of zaragozic acid to the biofilm formation assay of B. subtilis and S. aureus inhibited biofilm formation. Biofilm formation in B. subtilis is observed as a pellicle formed in the surface air–liquid of standing cultures, while S. aureus forms biofilms attached to the submerged surfaces (at the bottom of the well plate). Crystal violet staining was used in the S. aureus assay for better visualization. The effects of a range of drug concentration are shown.