Identification and characterization of the Bacillus subtilis spore germination protein GerY

Abstract

In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth when nutrients become available. To identify uncharacterized factors involved in the exit from dormancy, we performed a transposon-sequencing screen taking advantage of the loss of spore heat resistance that accompanies germination. We reasoned that transposon insertions that impair but do not block germination will lose resistance more slowly than wild type after exposure to nutrients and will therefore survive heat treatment. Using this approach, we identified most of the known germination genes and several new ones. We report an initial characterization of 15 of these genes and a more detailed analysis of one (ymaF). Spores lacking ymaF (renamed gerY) are impaired in germination in response to both L-alanine and L-asparagine, D-glucose, D-fructose, and K⁺. GerY is a soluble protein synthesized under σ^E control in the mother cell. A YFP-GerY fusion localizes around the developing and mature spore in a manner that depends on CotE and SafA, indicating that it is a component of the spore coat. Coat proteins encoded by the gerP operon and gerT are also required for efficient germination, and we show that spores lacking two or all three of these loci have more severe defects in the exit from dormancy. Our data are consistent with a model in which GerY, GerT, and the GerP proteins are required for efficient transit of nutrients through the coat to access the germination receptors, but each acts independently in this process.

Importance: Pathogens in the orders Bacillales and Clostridiales resist sterilization by differentiating into stress-resistant spores. Spores are metabolically inactive and can remain dormant for decades, yet upon exposure to nutrients, they rapidly resume growth, causing food spoilage, food-borne illness, or life-threatening disease. The exit from dormancy, called germination, is a key target in combating these important pathogens. Here, we report a high-throughput genetic screen using transposon sequencing to identify novel germination factors that ensure the efficient exit from dormancy. We identify several new factors and characterize one in greater detail. This factor, renamed GerY, is part of the proteinaceous coat that encapsulates the dormant spore. Our data suggest that GerY enables efficient transit of nutrients through the coat to trigger germination.

Keywords: coat; dormancy; germination; sporulation.

Fig 1

A genetic enrichment combined with transposon sequencing identifies new germination factors. (A) Schematic of the enrichment screen. A B. subtilis transposon library was sporulated in liquid medium. A sample was collected at the onset of starvation (not shown). The spore-containing culture was washed, divided into three, and incubated with ddH₂O, L-alanine, or AGFK to induce germination. After 60 min, the cultures were incubated at 80°C for 20 min to kill germinated spores and non-sporulated (vegetative) cells. The cultures were then plated on LB agar. Spores that did not germinate prior to heat-kill survived (light blue ovals) and formed colonies. Colonies from each condition were separately pooled. A fraction of the L-ala and AGFK mixtures was subjected to a second round of sporulation, germination, heat-kill, and plating (not shown). The transposon insertion sites were identified by deep sequencing and mapped to the B. subtilis 168 reference genome. (B) Transposon insertion profiles from five regions of the B. subtilis genome are depicted. Red boxes highlight gerD, ymaF, yodN, ydgB, and yqeF that were significantly enriched (P < 0.05) for transposon insertions after two rounds of sporulation and germination with L-ala and AGFK compared to water. Each vertical line indicates an insertion site, and the height represents the number of sequencing reads. The maximum number of reads shown is indicated in each panel. Additional insertion profiles can be found in Fig. S1.

Fig 2

YmaF, YodN, YdgB, and YqeF are required for efficient spore germination. Germination assays of wild-type (WT), ∆ymaF, ∆yodN, ∆ydgB, and ∆yqeF spores in response to L-alanine or AGFK as assessed by the percent reduction in OD₆₀₀ over time. Purified spores from the indicated strains were incubated with the germinant indicated at 37°C, and the drop in optical density was monitored over time. The germination defect of all four mutants could be complemented in trans. Representative data from one of three biological replicates are shown. Error bars indicate ±SD of three technical replicates.

Fig 3

YmaF, YodN, and YdgB are synthesized in the mother cell during sporulation. (A) Representative images of sporulating strains harboring the indicated yfp fusion to the promoters of ymaF, yodN, ydgB, and yqeF. The sporulation time point (in hours) that was visualized for each strain is indicated in the low right corner of the YFP image. A sporulation time course for each strain can be found in Fig. S5. (B) Representative images of sporulating cells harboring YFP fusions to YmaF or YdgB. The sporulation time point (in hours) that was visualized for each strain is indicated in the low right corner of the YFP image. The membranes (false-colored red) were stained with the fluorescent dye TMA-DPH and merged with the YFP signal (false-colored green). Scale bars indicate 2 µm.

Fig 4

GerY is a coat protein. (A) Germination assays with a range of L-alanine concentrations. Purified spores from the indicated strains were incubated with L-alanine at 37°C, and the drop in optical density was monitored over time. (B) Representative images of purified spores harboring fluorescent fusions to GerT, GerPA, and GerY. In the absence of SafA or CotE, YFP-GerY and GerT-GFP are not retained in the spore coat. Scale bar indicates 2 µm. Spores lacking the inner coat proteins CotP, CotD, CotF (C) or the outer coat proteins CotS, CotA, CotC (D) have mild germination defects compared to wild type and ∆gerT. Purified spores from the indicated strains were incubated with 1 mM L-alanine at 37°C, and the drop in optical density was monitored over time. Representative data from one of three biological replicates are shown in panels A, C, and D. Error bars indicate ±SD of three technical replicates.

Fig 5

GerY, GerT, and GerP function in different genetic pathways. (A) Germination assays of single, double, and triple mutants of gerY, gerT, and gerP suggest each factor functions separately to promote efficient germination. Purified spores from the indicated strains were incubated with 10 mM L-alanine at 37°C, and the drop in optical density was monitored over time. (B) Germination assays of ∆cotB in the presence and absence of gerT, gerP, or gerY suggest each factor functions separately to promote efficient germination. (C) Germination assays with spores lacking their coat indicate that the reduced germination efficiency of ∆gerT, ∆gerP, and ∆gerY spores results from defects in the spore coat. Purified spores with or without their coats were incubated with 10 mM L-alanine at 37°C, and the drop in optical density was monitored over time. Representative data from one of three biological replicates are shown. Error bars indicate ±SD of three technical replicates.

Fig 6

Schematic model for the role of GerY, GerT, and GerP proteins in the efficient transit of the germinants through the spore coat. Germinants (blue circles) must transit the crust, the outer and inner coat, the outer spore membrane, the cortex, and the germ cell wall to reach the germinant receptors in the inner spore membrane. GerY, GerT, and GerP are proposed to increase the porosity of the coat. In their absence, germinants are hindered in their access to the receptors.