Genetic Screens Identify Additional Genes Implicated in Envelope Remodeling during the Engulfment Stage of Bacillus subtilis Sporulation

Abstract

During bacterial endospore formation, the developing spore is internalized into the mother cell through a phagocytic-like process called engulfment, which involves synthesis and hydrolysis of peptidoglycan. Engulfment peptidoglycan hydrolysis requires the widely conserved and well-characterized DMP complex, composed of SpoIID, SpoIIM, and SpoIIP. In contrast, although peptidoglycan synthesis has been implicated in engulfment, the protein players involved are less well defined. The widely conserved SpoIIIAH-SpoIIQ interaction is also required for engulfment efficiency, functioning like a ratchet to promote membrane migration around the forespore. Here, we screened for additional factors required for engulfment using transposon sequencing in Bacillus subtilis mutants with mild engulfment defects. We discovered that YrvJ, a peptidoglycan hydrolase, and the MurA paralog MurAB, involved in peptidoglycan precursor synthesis, are required for efficient engulfment. Cytological analyses suggest that both factors are important for engulfment when the DMP complex is compromised and that MurAB is additionally required when the SpoIIIAH-SpoIIQ ratchet is abolished. Interestingly, despite the importance of MurAB for sporulation in B. subtilis, phylogenetic analyses of MurA paralogs indicate that there is no correlation between sporulation and the number of MurA paralogs and further reveal the existence of a third MurA paralog, MurAC, within the Firmicutes. Collectively, our studies identify two new factors that are required for efficient envelop remodeling during sporulation and highlight the importance of peptidoglycan precursor synthesis for efficient engulfment in B. subtilis and likely other endospore-forming bacteria. IMPORTANCE In bacteria, cell envelope remodeling is critical for cell growth and division. This is also the case during the development of bacteria into highly resistant endospores (spores), known as sporulation. During sporulation, the developing spore becomes internalized inside the mother cell through a phagocytic-like process called engulfment, which is essential to form the cell envelope of the spore. Engulfment involves both the synthesis and hydrolysis of peptidoglycan and the stabilization of migrating membranes around the developing spore. Importantly, although peptidoglycan synthesis has been implicated during engulfment, the specific genes that contribute to this molecular element of engulfment have remained unclear. Our study identifies two new factors that are required for efficient envelope remodeling during engulfment and emphasizes the importance of peptidoglycan precursor synthesis for efficient engulfment in the model organism Bacillus subtilis and likely other endospore-forming bacteria. Finally, our work highlights the power of synthetic screens to reveal additional genes that contribute to essential processes during sporulation.

Keywords: cell envelope; engulfment; morphogenesis; peptidoglycan; peptidoglycan remodeling; spores; sporulation.

FIG 1

Tn-seq reveals genes involved in PG synthesis and hydrolysis that are important for sporulation in the absence of spoIIB. (A) Schematic representation of normal (left) and abnormal (right) engulfment in a wild-type (WT) cell and ΔspoIIB mutant cell, respectively. In WT cells, the asymmetric septum curves and engulfment proceeds evenly around the forespore. In ΔspoIIB cells, the asymmetric septum bulges and protrudes into the mother cell. PG is shaded in gray. (B) Scatterplot showing fold reduction of transposon insertions in ΔspoIIB (bCR1560) relative to WT (bDR2413) cells with corresponding P values. Genes involved in PG synthesis (murAB, murJ) and hydrolysis (yrvJ) with high fold reduction in ΔspoIIB compared to WT cells and a low P value are labeled and colored cyan. (C) Tn-seq profiles at the yrvJ, murJ, and murAB genomic loci of WT (bDR2413) and ΔspoIIB (bCR1560) cells following 30 h of growth and sporulation in exhaustion medium. The height of the vertical lines represents the number of Tn-seq reads at each position. Shaded regions highlight the significant reduction in sequencing reads at yrvJ, murJ, and murAB loci.

FIG 2

Engulfment initiation and progression in spoIIB and yrvJ mutants. (A) Engulfment initiation and progression in the wild-type (WT) (bAT68), ΔyrvJ (bHC175), ΔspoIIB (bHC180), and ΔspoIIB ΔyrvJ (bHC176) strains at 2 h (T2) and 3 h (T3) after onset of sporulation. Forespore cytoplasm was visualized using a forespore reporter (P_spoIIQ-cfp [false-colored cyan in merged images]). Cell membranes were visualized with TMA-DPH fluorescent membrane dye and are false-colored red in merged images. Scale bar = 2 μm. (B) Average sporulation efficiency (mean percentage ± standard deviation [SD]; n = 3) of the ΔyrvJ (bAT144), ΔspoIIB (bCR1560), and ΔspoIIB ΔyrvJ (bAT152) mutant strains as a percentage of the WT (bDR2413). Error bars represent SD from three biological replicates. (C) Average frequency (mean percentage ± SD; n = 3) of cells that had completed engulfment during a sporulation time course in WT (bAT68 [blue]) and ΔyrvJ (bHC175 [green]) cells, plotted on a nonlogarithmic scale (n > 300 per time point, per strain, per replicate). Error bars represent SD from three biological replicates. (D) Average frequency (mean percentage ± SD; n = 3) of sporulating cells containing flat (blue), bulging (green), abnormal (red), and even (yellow) septa during a sporulation time course in WT (bAT68), ΔyrvJ (bHC175), ΔspoIIB (bHC180), and ΔspoIIB ΔyrvJ (bHC176) cells (n > 300 per time point, per strain, per replicate). Error bars represent SD from three biological replicates. Representative images of cells containing each of the septal phenotypes are shown in Fig. S5. (E) Average frequency (mean percentage ± SD; n = 3) of cells that had completed engulfment during a sporulation time course in WT (bAT68 [blue]), ΔyrvJ (bHC175 [green]), ΔspoIIB (bHC180 [red]), and ΔspoIIB ΔyrvJ (bHC176 [yellow]) cells (n > 300 per time point, per strain, per replicate). Error bars represent SD from three biological replicates. *, P < 0.01 by Student's t test performed on the mean of replicates (n = 3) at T4 for the ΔspoIIB mutant versus ΔspoIIB ΔyrvJ mutant.

FIG 3

Engulfment initiation and progression in spoIIB and murAB mutants. (A) Engulfment initiation and progression in the wild-type (WT) (bAT344), ΔmurAB (bHC217), ΔspoIIB (bHC216), and ΔspoIIB ΔmurAB (bHC329) strains at 2 h (T2) and 3 h (T3) after onset of sporulation. Forespore cytoplasm was visualized using a forespore reporter (P_spoIIQ-gfp [false-colored cyan in merged images]). Cell membranes were visualized with TMA-DPH fluorescent membrane dye and are false-colored red in merged images. Scale bar = 2 μm. (B) Average sporulation efficiency (mean percentage ± SD; n = 3) of the ΔmurAB (bAT73), ΔspoIIB (bCR1560), and ΔspoIIB ΔmurAB (bHC203) mutant strains as a percentage of the WT (bDR2413). Error bars represent SD from three biological replicates. (C) Average frequency (mean percentage ± SD; n = 3) of cells that had completed engulfment during a sporulation time course in WT (bAT344 [blue]) and ΔmurAB (bHC217 [green]) cells, plotted on a nonlogarithmic scale (n > 300 per time point, per strain, per replicate). Error bars represent SD from three biological replicates. *, P < 0.0001 by Student's t test performed on the mean of replicates (n = 3) at T4 for the WT versus the ΔmurAB mutant. (D) Average frequency (mean percentage ± SD; n = 3) of sporulating cells containing flat (blue), bulging (green), abnormal (red), and even (yellow) septa during a sporulation time course in WT (bAT344), ΔmurAB (bHC217), ΔspoIIB (bHC216), and ΔspoIIB ΔmurAB (bHC329) cells (n > 300 per time point, per strain, per replicate). Error bars represent SD from three biological replicates. Representative images of cells containing each of the septal phenotypes are shown in Fig. S3. (E) Average frequency (mean percentage ± SD; n = 3) of cells that had completed engulfment during a sporulation time course in WT (bAT344 [blue]), ΔmurAB (bHC217 [green]), ΔspoIIB (bHC216 [red]), and ΔspoIIB ΔmurAB (bHC329 [yellow]) cells (n > 300 per time point, per strain, per replicate). Error bars represent SD from three biological replicates.

FIG 4

GFP-SpoIIP localization in the absence of spoIIB and murAB. (A) GFP-SpoIIP localization and (B) distribution of GFP-SpoIIP mean fluorescence intensity (a.u.) in the wild-type (WT) (bHC544), ΔmurAB (bHC545), ΔspoIIB (bHC546), and ΔspoIIB ΔmurAB (bHC547) strains at 2.5 h after onset of sporulation (T2.5). GFP signal is false-colored cyan in merged images. Cell membranes were visualized with TMA-DPH fluorescent membrane dye and are false-colored red in merged images. Scale bar = 2 μm. Panel B is a superplot with totals of 489, 528, 645, and 694 cells for the WT, ΔmurAB, ΔspoIIB, and ΔspoIIB ΔmurAB strains, respectively. *, P < 0.01, and **, P < 1 × 10⁻⁸, by Kolmogorov-Smirnov test performed on the combined distribution of replicates (n = 3). The Welch’s t test was also performed on the means with P < 0.05 for the WT versus the ΔmurAB mutant and the ΔspoIIB mutant versus the ΔspoIIB ΔmurAB mutant. (C) Immunoblot analysis of SpoIIP in the ΔspoIIP (bHC550), WT (bDR2413), ΔmurAB (bAT73), ΔspoIIB (bCR1560) and ΔspoIIB ΔmurAB (bHC203) strains at T2.5. SpoIIP was detected using anti-SpoIIP antibodies. Spo0J was used as a loading control and was detected using anti-Spo0J antibodies. (D) Mean integrated density of SpoIIP bands relative to the WT as detected by immunoblot analysis in panel C. Error bars represent the SD from three biological replicates. *, P < 0.0001 by Student's t test.

FIG 5

Tn-seq reveals genes involved in PG synthesis and hydrolysis that are important for sporulation in the absence of spoIIIAH. (A) Schematic illustration of an engulfing B. subtilis cell (left), highlighting the protein interaction between SpoIIQ (green) in the forespore membrane and SpoIIIAH (blue) in the engulfing mother cell membrane. The SpoIIQ-SpoIIIAH interaction is required for efficient A-Q complex formation, engulfment, coat assembly, germination, and σ^K activation. PG is shaded in light gray. (B) Scatterplot showing fold-reduction of transposon insertions in ΔspoIIIAH (bCR1117) relative to WT (bDR2413) cells with corresponding P values. Genes related to peptidoglycan synthesis (murAB, murJ, pbpF), metabolism (yxeR) and coat assembly (cotE) with high fold reduction in ΔspoIIIAH compared to WT cells and a low P value are labeled and colored cyan. (C) Tn-seq profiles at the pbpF, murJ, and murAB genomic loci of WT (bDR2413) and ΔspoIIIAH (bCR1117) cells, following 30 h of growth and sporulation in exhaustion medium. Height of vertical lines represents number of Tn-seq reads at each position. Shaded regions highlight the significant reduction in sequencing reads at the pbpF, murJ, and murAB loci.

FIG 6

Engulfment initiation and progression in spoIIIAH and murAB mutants. (A) Engulfment initiation and progression in the wild-type (WT) (bKH3), ΔspoIIIAH (bKH4), ΔmurAB (bKH5), and ΔspoIIIAH ΔmurAB (bKH6) strains at 2 h (T2), T3, T3.5, and T4 after onset of sporulation. Cell membranes were visualized with TMA-DPH fluorescent membrane dye. Scale bar = 2 μm. (B) Average sporulation efficiency (mean percentage ± SD; n = 3) of the ΔmurAB (bAT73), ΔspoIIIAH (bKH7), and ΔspoIIIAH ΔmurAB (bKH40) mutant strains as a percentage of the WT (bDR2413). Error bars represent SD from three biological replicates. (C) Average frequency (mean percentage ± SD; n = 3) of cells that had completed engulfment during a sporulation time course in WT (bKH3 [blue]), ΔmurAB (bKH5 [green]), ΔspoIIIAH (bKH4 [red]), and ΔspoIIIAH ΔmurAB (bKH6 [yellow]) cells (n > 100 per time point, per strain, per replicate). Error bars represent SD from three biological replicates. *, P < 0.01 by Student's t test performed on the mean of replicates (n = 3) at T4 for the WT versus the ΔmurAB mutant, the WT versus the ΔspoIIIAH mutant, and the WT versus the ΔspoIIIAH ΔmurAB mutant.

FIG 7

Phylogenetic analysis of MurA homologs. (A) Phylogenetic mapping of MurA (in gray) and Spo0A_C as a marker of sporulation (in red) on a maximum likelihood reference phylogeny of the Firmicutes based on a supermatrix containing 497 taxa with 3,776 amino acid positions and inferred with IQ-TREE 1.6.3 using LG+I+G4. Gray dots correspond to supports higher than 80%, and the scale bar corresponds to the average number of substitutions per site. The presence of Spo0A_C homologs is indicated in red in front of each tip. The presence of MurA homologs is indicated with gray bars, and the length of the bar corresponds to the number of paralogs, which varies from 1 to 4. (B) Maximum likelihood phylogenetic tree of MurA homologs from the Firmicutes based on an alignment of 938 taxa and 283 amino acid positions and inferred with IQ-TREE using LG+R10. For clarity, only node supports higher than 80% are displayed. The scale bar corresponds to the average number of substitutions per site. A high-resolution version of this figure can be found at https://doi.org/10.6084/m9.figshare.20464098.v1.