Characterization and subcellular localization of a bacterial flotillin homologue

Abstract

The process of endospore formation in Bacillus subtilis is complex, requiring the generation of two distinct cell types, a forespore and larger mother cell. The development of these cell types is controlled and regulated by cell type-specific gene expression, activated by a s-factor cascade. Activation of these cell type-specific sigma factors is coupled with the completion of polar septation. Here, we describe a novel protein, YuaG, a eukaryotic reggie/flotillin homologue that is involved in the early stages of sporulation of the Gram-positive model organism B. subtilis. YuaG localizes in discrete foci in the membrane and is highly dynamic. Purification of detergent-resistant membranes revealed that YuaG is associated with negatively charged phospholipids, e.g. phosphatidylglycerol (PG) or cardiolipin (CL). However, localization of YuaG is not always dependent on PG/CL in vivo. A yuaG disruption strain shows a delay in the onset of sporulation along with reduced sporulation efficiency, where the spores develop to a certain stage and then appear to be trapped at this stage. Our results indicate that YuaG is involved in the early stage of spore development, probably playing a role in the signalling cascade at the onset of sporulation.

Functional microdomains in bacterial membranes

“…While no exact function was ascribed to these bacterial proteins, a B. subtilis mutant lacking the flotillin-like protein showed a delay in the onset of sporulation and reduced sporulation efficiency (Donovan and Bramkamp 2009). Attempts to determine the type of lipid that led to the punctate distribution of the B. subtilis flotillin-like protein were inconclusive, finding only that its localization was not dependent on lipids containing phosphatidylglycerol or cardiolipin (Donovan and Bramkamp 2009). Cardiolipin was of particular interest, because it had been shown to occur in patches in the B. subtilis membrane (Kawai et al 2004;Matsumoto et al 2006;Mileykovskaya and Dowhan 2009).…”

“…Interestingly, bioinformatic analyses indicate that most bacterial genomes encode proteins whose amino acid sequences show some similarity to Flotillin-1. While these bacterial proteins have not been extensively investigated, two reports (Zhang et al 2005;Donovan and Bramkamp 2009) demonstrated that the flotillin-like proteins from the spore-forming Bacillus halodurans and Bacillus subtilis are distributed heterogeneously in the cytoplasmic membrane displaying a punctate pattern along the entire cell. While no exact function was ascribed to these bacterial proteins, a B. subtilis mutant lacking the flotillin-like protein showed a delay in the onset of sporulation and reduced sporulation efficiency (Donovan and Bramkamp 2009).…”

“…While these bacterial proteins have not been extensively investigated, two reports (Zhang et al 2005;Donovan and Bramkamp 2009) demonstrated that the flotillin-like proteins from the spore-forming Bacillus halodurans and Bacillus subtilis are distributed heterogeneously in the cytoplasmic membrane displaying a punctate pattern along the entire cell. While no exact function was ascribed to these bacterial proteins, a B. subtilis mutant lacking the flotillin-like protein showed a delay in the onset of sporulation and reduced sporulation efficiency (Donovan and Bramkamp 2009). Attempts to determine the type of lipid that led to the punctate distribution of the B. subtilis flotillin-like protein were inconclusive, finding only that its localization was not dependent on lipids containing phosphatidylglycerol or cardiolipin (Donovan and Bramkamp 2009).…”

Connection of KinC to flotillins and potassium leakage in Bacillus subtilis

“…First, it is attributed to us that the floTA mutant phenocopies the kinC mutant, but we have actually published that they have different sporulation efficiencies (Yepes et al, 2012). In addition, it is attributed to us that the flotillin-deficient mutant shows reduced sporulation, but this observation was initially reported by another laboratory (Donovan & Bramkamp, 2009) and later confirmed by us (Yepes et al, 2012). Finally, the localization of KinC in a single membrane focus was also attributed to us.…”

Controlled Assembly of SNAP–PNA–Fluorophore Systems on DNA Templates To Produce Fluorescence Resonance Energy Transfer

“…Examples include: localization and trafficking of a fusion protein in the cell membrane, labeling of antibody fragments, designing fluorescent biosensors, controlling yeast transcription and visualization of metabolite signaling. [49][50][51][52][53][54][55][56] Scheme 1: Coupling of BG-PNA-Atto dyes to the SNAP protein to produce a thioether bond between cysteine of SNAP and benzyl linker of modified construct.…”