Genetic Evidence for Signal Transduction within the Bacillus subtilis GerA Germinant Receptor

Abstract

Bacterial spores can rapidly exit dormancy through the process of germination. This process begins with the activation of nutrient receptors embedded in the spore membrane. The prototypical germinant receptor in Bacillus subtilis responds to l-alanine and is thought to be a complex of proteins encoded by the genes in the gerA operon: gerAA, gerAB, and gerAC. The GerAB subunit has recently been shown to function as the nutrient sensor, but beyond contributing to complex stability, no additional functions have been attributed to the other two subunits. Here, we investigate the role of GerAA. We resurrect a previously characterized allele of gerA (termed gerA*) that carries a mutation in gerAA and show that it constitutively activates germination even in the presence of a wild-type copy of gerA. Using an enrichment strategy to screen for suppressors of gerA*, we identified mutations in all three gerA genes that restore a functional receptor. Characterization of two distinct gerAB suppressors revealed that one (gerAB[E105K]) reduces the GerA complex's ability to respond to l-alanine, while another (gerAB[F259S]) disrupts the germinant signal downstream of l-alanine recognition. These data argue against models in which GerAA is directly or indirectly involved in germinant sensing. Rather, our data suggest that GerAA is responsible for transducing the nutrient signal sensed by GerAB. While the steps downstream of gerAA have yet to be uncovered, these results validate the use of a dominant-negative genetic approach in elucidating the gerA signal transduction pathway. IMPORTANCE Endospore formers are a broad group of bacteria that can enter dormancy upon starvation and exit dormancy upon sensing the return of nutrients. How dormant spores sense and respond to these nutrients is poorly understood. Here, we identify a key step in the signal transduction pathway that is activated after spores detect the amino acid l-alanine. We present a model that provides a more complete picture of this process that is critical for allowing dormant spores to germinate and resume growth.

Keywords: exit from dormancy; germination; nutrient receptor; sporulation.

FIG 1

GerA* triggers DPA release and SleB activation. (A) Phase-contrast micrographs of the indicated strains sporulated by nutrient exhaustion for 30 h. gerA⁺/gerA⁺ is a merodiploid strain with two copies of the wild-type (WT) gerA locus. gerA⁺/gerA* is a merodiploid strain with a wild-type gerA locus and a mutant gerA* operon at an ectopic genomic locus. Experiments were performed in biological triplicate; representative images are shown. Bar, 2 μm. (B) Sporulated cultures in panel A were heat treated (80°C for 20 min), and serial dilutions were plated on LB to assess heat-resistant CFU. Wild-type spore viability (3.3 × 10⁸ CFU/mL) was set to 100%. Δs Δc, ΔsleB ΔcwlJ. The error bars indicate the standard deviation (n = 3). (C) Phase-gray and phase-bright spores were purified from sporulated cultures in panel A. Spores were boiled to release dipicolinic acid (DPA). DPA was then quantified using TbCl₃ compared to standards. The values are reported as micrograms of DPA released from 1 mL of purified spores adjusted to OD₆₀₀ = 1. ΔVFA, ΔspoVFA. The error bars indicate the standard deviation (n = 4).

FIG 2

An enrichment screen for suppressors of gerA*. (A) Cultures were sporulated by nutrient exhaustion and heat treated (80°C for 20 min). Serial dilutions were plated on LB to assess heat-resistant CFU. Wild-type spore viability (3.0 × 10⁸ CFU/mL) was set to 100%. Δ5, ΔgerA ΔgerBB ΔgerKB ΔyndE ΔyfkT. The values are reported on a logarithmic scale for clarity. The error bars indicate the standard deviation (n = 5). (B) Cultures of Δ5 gerA* cells were sporulated by nutrient exhaustion and heat treated. A sample was taken to assess heat-resistant CFU by plating serial dilutions on LB. Another sample of ∼10⁶ viable spores was then used to inoculate fresh cultures, which allowed the spores to germinate, outgrow, and resporulate upon nutrient exhaustion. This process was repeated until spore viability increased. A sample of eight independent lineages are shown. The values are reported on a linear scale for clarity. (C) Cultures were sporulated overnight and heat treated. Serial dilutions were plated on LB to assess heat-resistant CFU. Δ5 gerA⁺ spore viability (2.2 × 10⁸ CFU/mL) was set to 100%. The error bars indicate the standard deviation (n = 3). (D) Micrographs of sporulated cultures from panel C. Representative images from three biological replicates are shown. Bar, 2 μm.

FIG 3

Analysis of GerAB suppressor mutants. (A) The indicated strains were sporulated overnight and heat treated. Serial dilutions were plated on LB to assess heat-resistant CFU. Δ4, ΔgerBB ΔgerKB ΔyndE ΔyfkT. gerAB⁺ spore viability (3.5 × 10⁸ CFU/mL) was set to 100%. The error bars indicate the standard deviation (n = 3). (B) Immunoblots of gerAB variants. Cultures were sporulated overnight. Phase-gray and phase-bright spores were purified using lysozyme and SDS. The spores were physically disrupted, and lysates were subjected to SDS-PAGE followed by immunoblot analysis to detect the presence of GerAA. SleB and SigA were analyzed to control for loading. Representative immunoblots are shown (n = 3). (C, D) Germination of gerAB variants. Cultures were sporulated overnight, and phase-bright spores were purified using density gradients. Spores were resuspended in 96-well plates in the presence or absence of l-alanine and agitated for 8 h at 37°C. Optical density at time zero was normalized to 1, and subsequent measurements were taken every 2 min. The shaded area indicates the standard deviation of three biological replicates. AB(var) indicates gerAB(E105K) or gerAB(F259S) in panel C or D, respectively. Note that the solid purple, dotted purple, and dotted black lines are superimposed. OD₆₀₀, optical density at 600 nm.

FIG 4

Epistatic analysis of gerAB(F259S) and gerAB(T287L). (A) Cultures were sporulated by nutrient exhaustion. Representative phase-contrast micrographs of three biological replicates are shown. Bar, 2 μm. The cultures were heat treated (80°C for 20 min), serially diluted, and plated on LB to assess heat-resistant CFU. gerAB⁺ spore viability (3.7 × 10⁸ CFU/mL) was set to 100%. (B) Model of signal transduction within the GerA complex. GerAC has been omitted for clarity.

FIG 5

Predicted structures of GerAA and GerAB. (A) The top panel shows Alphafold2-predicted structures of GerAA and GerAB in complex, situated in the inner spore membrane (gray). The bottom panel shows the predicted structures rotated 90° for a top-down view. Helix 4 of GerAB and helix 6 of GerAA are predicted to form a contact interface between the two proteins. The binding pocket of GerAB is visible, as is the proximity of the flexible loop in GerAB and the perpendicular helix in GerAA. (B) View of the predicted interface between the flexible loop of GerAB and the perpendicular helix of GerAA. Residue F259 of GerAB is shown in red, and residue P326 of GerAA is shown in green. Other residues that, when mutated, were able to suppress gerAA(P326S) are highlighted in red. (C) View of the l-alanine-binding pocket in GerAB. Residue E105 is shown in red. Residues that have been previously shown to constitute the binding pocket (V101, T287, L199, and Y291) are shown in green (12).