Developmentally regulated proteolysis by MdfA and ClpCP mediates metabolic differentiation during Bacillus subtilis sporulation

Abstract

Bacillus subtilis sporulation entails a dramatic transformation of the two cells required to assemble a dormant spore, with the larger mother cell engulfing the smaller forespore to produce the "cell within a cell" structure that is a hallmark of endospore formation. Sporulation also entails metabolic differentiation, whereby key metabolic enzymes are depleted from the forespore but maintained in the mother cell. This reduces the metabolic potential of the forespore, which becomes dependent on mother cell metabolism and the SpoIIQ-SpoIIIA channel to obtain metabolic building blocks necessary for development. We demonstrate that metabolic differentiation depends on the ClpCP protease and a forespore-produced protein encoded by the yjbA gene, which we have renamed MdfA (metabolic differentiation factor A). MdfA is conserved in aerobic endospore formers and required for spore resistance to hypochlorite. Using mass spectrometry and quantitative fluorescence microscopy, we show that MdfA mediates the depletion of dozens of metabolic enzymes and key transcription factors from the forespore. An accompanying study by Massoni and colleagues demonstrates that MdfA is a ClpC adaptor protein that directly interacts with and stimulates ClpCP activity. Together, these results document a developmentally regulated proteolytic pathway that reshapes forespore metabolism, reinforces differentiation, and enhances spore resistance to the oxidant hypochlorite.

Keywords: metabolic reprogramming; metabolism; oxidative stress; proteolysis; sporulation.

Figure 1.

Metabolic reprogramming during spore formation. (A) B. subtills sporulation. (I) Vegetative cell. (II) Polar septation. (III) Engulfment. (IV) Spore maturation. (V) Mother cell lysis and spore release. Sporulation-specific σ factors are shown in the cells and stages in which they are active. (B) Intercellular nurturing during B. subtilis sporulation. Mother cell TCA cycle and amino acid synthesis (AAs; colored circles) enable forespore protein synthesis. Metabolites are transported via the SpoIIQ–SpoIIIA (Q-A) channel. (Blue) SpoIIQ, (red) SpoIIIAA-AH. (C) Fluorescence micrographs of sporulating cells with FM 4-64-stained membranes (red) and GFP fusions (green) to proteins involved in transcription (RpoZ), TCA cycle (Icd and CitZ), nucleotide biosynthesis (GuaB), and amino acid biosynthesis (ArgH, LeuD, and MetE). Arrowheads indicate representative forespores. Scale bar, 1 µm. (D) Violin plots showing the ratios of the mean GFP fluorescence in the forespore to that in the mother cell (FS:MC) for the proteins in C. Each green dot represents the fluorescent ratio of an individual sporangium. The black dotted lines delimit the middle 75% of the data, and the black solid lines indicate the median. The blue dashed line indicates equal intensity in both cells, and the red dashed line indicates a fluorescence ratio of 0.25, as predicted for protein dilution due to increased forespore volume and decreased mother cell volume (Supplemental Fig. S1). Values <0.25 likely entail protein depletion. (E) Diagram showing the two pathways, dilution due to forespore growth, and a specific protein depletion pathway.

Figure 2.

Forespore growth mediates protein dilution. (A) Visualization of CitZ-GFP, Icd-GFP, and GFP produced from the citZ promoter (PcitZ-GFP) in sporulating cells containing (SpoIIQ⁺) or lacking (SpoIIQ⁻) SpoIIQ. Arrowheads indicate forespores. Membranes were stained with FM 4-64 (red). (B) Quantification of the forespore to mother cell (FS:MC) GFP ratio for the strains shown in A. The “+” and “−” symbols on the X-axis indicate the presence and absence of SpoIIQ, respectively. Dashed lines indicate the middle 75% of the data, and solid lines indicate the median. (C) Time-lapse fluorescence microscopy of FM 4-64-stained sporulating cells producing CitZ-GFP (top) or Icd-GFP (bottom) in the presence of 30 µg/mL cerulenin or an equal volume of solvent. Forespores are indicated by arrowheads in the last image at the right. (D) Quantification of the FS:MC GFP ratio of sporulating cells from the time lapses shown in B over time, relative to the initial signal at t₀. Values represent the mean ± SD. Scale bars, 1 µm.

Figure 3.

Identification of the proteolytic pathway that mediates depletion of CitZ-GFP in the forespore. (A) Visualization of CitZ-GFP (green) in mutant sporulating cells lacking the indicated proteases. Membranes were stained with FM 4-64 (red). Scale bar, 1 µm. (B) Log₂ of the fold change in the FS:MC GFP ratio for each strain relative to wild type. ClpX and ClpP mutants failed to sporulate, so it was not possible to calculate the CitZ-GFP fluorescence ratios (NA). More than 100 cells were analyzed for each strain. (C) The newly synthesized polar septum traps the forespore chromosome (blue), with the origin of replication (oriC; green circle) in the forespore and the terminus (ter; red circle) in the mother cell. The remaining forespore chromosome is translocated into the forespore by SpoIIIE (IIIE). The diagram at the right shows the chromosome, and highlights the region initially trapped in the forespore (gray). IIIE is required to move genes in the unshaded region into the forespore. (D) Visualization of CitZ-GFP in sporulating cells lacking σ^F (ΔsigF), lacking σ^E (ΔsigE), containing a SpoIIIE allele unable to hydrolyze ATP (IIIE^ATPase), lacking MdfA (ΔmdfA), lacking ClpC (ΔclpC), lacking both MdfA and ClpC (ΔmdfA ΔclpC), containing the MdfA E116K allele (mdfA^E116K clpC^WT), or containing the MdfA E116K and the ClpC K85E alleles (mdfA^E116K clpC^K85E). Membranes were stained with FM 4-64 (red). (E) Log₂ of the FS:MC fluorescence ratio of each strain shown in D and of mutants lacking selected σ^F-regulated genes initially trapped in the mother cell (ori-distal σ^F-reg. genes). Every dot represents the log₂ of the average ratio of a different strain, color-coded according to the key at the right. More than 100 cells were analyzed for each strain. (F) Localization of MdfA-GFP. Membranes were stained with FM 4-64 (red). Scale bars, 1 µm.

Figure 4.

MdfA protects spores from oxidative damage. (A) Phylogenetic tree of Bacillus pumilus (Bp), Bacillus licheniformis (Bl), B. subtilis (Bs), Bacillus cereus (Bc) Staphylococcus aureus (Sa), and Clostridium perfringens (Cp). The mdfA (yellow) region is shown for every species, with each arrow representing a gene. Other ClpC adaptor proteins include mecA (orange) and spxH (red). (B) Sunburst diagram from PFAM representing the distribution of MdfA homologs across different taxa, with the majority being found in the Bacilli (blue), and with a few in viruses (gray), Mollicutes (purple), and Clostridia (orange). (C) Sporulating cultures of the wild type and the ΔmdfA mutant at t₃, t₆, and t₂₄ after resuspension. Membranes were stained with FM 4-64 (red) and MitotrackerGreen (green) for t₃ samples to discriminate between partially engulfed forespores (orange) and fully engulfed spores (green). Phase contrast images are shown for t₆ and t₂₄. (D) Relative spore titers of the wild-type (blue) and ΔmdfA (orange) strains after nutrient exhaustion and treatment with chloroform, ethanol (EtOH), heat, lysozyme, or NaOCl. Wild-type titers were normalized to 100%. Values represent the mean ± SD of three independent experiments. (E) Phase contrast time-lapse microscopy showing germination and outgrowth of purified spores from the wild-type and ΔmdfA strains treated with 0.25% NaOCl for 18 min or untreated. Images were acquired every 30 min for 3.5 h. (F) Quantification of the fraction of phase-bright (gray), germinated (blue), and outgrowing (orange) spores during time-lapse experiments. More than 100 spores were analyzed for each strain and treatment. Scale bars, 1 µm.

Figure 5.

Identification of proteins depleted in an MdfA-dependent manner. (A) Strategy to identify proteins depleted from the spore in an MdfA-dependent manner. Non-MdfA substrates (gray circles) are present in similar amounts in both MdfA⁺ and MdfA⁻ spores. MdfA substrates (green) are more abundant in MdfA⁻ than in MdfA⁺ spores. (B) Volcano plot of mass spectrometry results comparing protein abundance in MdfA⁻ versus MdfA⁺ spores when sporulation was induced by resuspension (see the Materials and Methods). Proteins with more than one log₂ fold change and >20 significance were considered potential substrates (blue dots). One-hundred-seventeen hits were observed (Supplemental Data File; raw data available via ProteomeXchange [Vizcaíno et al. 2013], identifier PXD051727). (C) Gene ontology analysis of the MdfA substrates identified through proteomics based on PANTHER results (Supplemental Data File). The expected percentage of proteins in the B. subtilis proteome belonging to each of the indicated pathways is in blue. The actual percentage of proteins in each pathway observed in the subset of proteins enriched in the absence of MdfA is in orange. The pathways displayed in the graph are overrepresented in the subset of proteins enriched in the absence of MdfA compared with their expected distribution in the whole proteome (Supplemental Data File). (D) Examples of validation using GFP fusions (full set in Supplemental Fig. S8). GFP fusion proteins (green) were visualized in MdfA⁺ (+) and MdfA⁻ (−) sporulating cells stained with FM 4-64 (red). Arrowheads indicate forespores. Scale bar, 1 µm. (E) Forespore (FS) GFP fluorescence relative to the median fluorescence of vegetative cells (VCs) present in the same sporulating cultures of strains carrying GFP fusions to the indicated proteins in MdfA⁺ (green) and MdfA⁻ (magenta) strains. The black dots indicate the median of each series; every green and magenta circle represents the relative fluorescence of an individual forespore. More than 150 forespores were analyzed for each protein (except for ThrC, where 23 and 44 forespores were analyzed in MdfA⁺ and MdfA⁻ backgrounds, respectively). The horizontal dashed line marks the relative forespore fluorescence value of 0.3, which is expected if vegetative proteins were only diluted by forespore growth. Fusions are sorted according to the fold increase in FS:VC fluorescence ratio in MdfA⁻ background compared with MdfA⁺ background, arranged from highest to lowest.

Figure 6.

Model of MdfA-dependent metabolic differentiation. (A) MdfA (orange) is produced in the forespore under σ^F control, interacts with ClpC (blue), and delivers target proteins (green) to ClpP (purple) for proteolysis. (B) In vegetative cells, ClpC and ClpP participate in protein quality control. During sporulation, MdfA is produced in the forespore, delivering target proteins to ClpCP for degradation and reducing the metabolic capacity of the forespore (green shading), which becomes dependent on the mother cell for metabolic precursors for biosynthesis.