Structural and Functional Analysis of the GerD Spore Germination Protein of Bacillus Species

Li, Yunfeng; Jin, Kai; Ghosh, Sumana; Devarakonda, P.; Carlson, Kristina; Davis, Andrew John; Stewart, Kerry-Ann V.; Cammett, Elizabeth; Rossi, Patricia Pelczar; Setlow, Barbara; Lu, Min; Setlow, Peter; Hao, Bing

doi:10.1016/j.jmb.2014.02.004

Cited by 21 publications

(28 citation statements)

References 45 publications

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“…Similarly, insertions in the gerK operon were largely absent in the Δ4 gerK + library. Consistent with the idea that the germinant receptors do not work with pathway‐specific signaling proteins that are critical for germination (Li et al ., ), there were no additional genes in which transposons were specifically under‐represented in one but not the other library. However, examination of the transposon insertion profiles identified an unanticipated set of genes in which insertions were over‐represented in the Δ4 gerK + library compared to the wild‐type and Δ4 gerA + libraries (Fig.…”

Section: Resultsmentioning

confidence: 92%

The Bacillus subtilis germinant receptor GerA triggers premature germination in response to morphological defects during sporulation

Ramírez-Guadiana

Meeske

Wang

et al. 2017

Molecular Microbiology

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During sporulation in Bacillus subtilis, germinant receptors assemble in the inner membrane of the developing spore. In response to specific nutrients these receptors trigger germination and outgrowth. In a transposon-sequencing screen, we serendipitously discovered that loss of function mutations in the gerA receptor partially suppress the phenotypes of >25 sporulation mutants. Most of these mutants have modest defects in the assembly of the spore protective layers that are exacerbated in the presence of a functional GerA receptor. Several lines of evidence indicate that these mutants inappropriately trigger activation of GerA during sporulation resulting in premature germination. These findings led us to discover that up to 8% of wild-type sporulating cells trigger premature germination during differentiation in a GerA-dependent manner. This phenomenon was observed in domesticated and undomesticated wild-type strains sporulating in liquid and on solid media. Our data indicate that the GerA receptor is poised on a knife's edge during spore development. We propose that this sensitized state ensures a rapid response to nutrient availability, but also elicits premature germination of spores with improperly assembled protective layers resulting in the elimination of even mildly defective individuals from the population.

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Section: Resultsmentioning

confidence: 92%

The Bacillus subtilis germinant receptor GerA triggers premature germination in response to morphological defects during sporulation

Ramírez-Guadiana

Meeske

Wang

et al. 2017

Molecular Microbiology

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“…Unfortunately, there are no data on the precise conditions in this compartment of sporulating cells. GerD is a helical trimer in vitro and may be capable of forming even higher-order oligomeric structures (11). In addition, GerD-GFP can form germinosome-like foci in dormant spores even in the absence of GRs (9).…”

Section: Figmentioning

confidence: 99%

“…Spores lacking GerD germinate normally with nonnutrient germinants that bypass the GRs, suggesting that GerD is required for efficient signal transduction from the nutrient-GR complex; however, the exact signaling mechanism remains unknown (10). The crystal structure of two-thirds of Geobacillus stearothermophilus GerD was solved recently, and the protein forms a rod-like trimer that is largely hydrophilic with a hydrophobic lipid anchor (11). Interestingly, a previous study as well as very recent work indicate that levels of GerD decrease markedly in the IM fraction from germinating and early outgrow-ing spores, suggesting that the protein becomes soluble or is degraded during this period (12,13).…”

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confidence: 99%

Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

et al. 2014

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Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the "germinosome," and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with >75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ϳ50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ϳ2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds. Spores of Bacillus subtilis are metabolically dormant and resistant to harsh environmental conditions, allowing them to survive for many years (1). However, the presence of specific nutrients triggers the process of germination, in which spores lose their dormancy and resistance and then outgrow into vegetative cells. Normally, for germination to occur, nutrients must cross the coat, outer membrane, cortex, and germ cell wall of the dormant spore to reach nutrient germinant receptors (GRs) located in the spore's inner membrane (IM) (1-3). Lipid molecules in the B. subtilis spore IM are largely (ϳ70%) immobile, but upon completion of spore germination, the volume encompassed by the IM expands ϳ1.5-fold, and the amount of the immobile lipid fraction decreases to ϳ25%, similar to the value for vegetative cells, all in the absence of detectable ATP synthesis (4). Binding of specific germinants to their cognate GRs leads to the release of the spore core's large store (ϳ25% of core dry weight) of dipicolinic acid (DPA) chelated to divalent cations, mostly Ca 2ϩ (CaDPA); CaDPA release is accompanied by some water uptake into the core and some loss of spore resistance. CaDPA release also triggers the degradation of the spore's peptidoglycan (PG) cortex by cortexlytic enzymes (CLEs), allowing for core expansion, further core water uptake, completion of germination, and, ultimately, outgrowth to gen...

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“…So far, the exact functions of the individual GR subunits and their interactions have not been elucidated (12). GRs are thought to cluster in complexes, so-called germinosomes, involving the GerD lipoprotein that influences GR-dependent germination rates (16,17) and possibly the SpoVA channels (located in the spore inner membrane) that are involved in the release of small molecules, mainly dipicolinic acid (DPA) and monovalent cations, and uptake of water during germination (12). Recently, it was shown that heat resistance and the germination rate of B. subtilis spores could be attributed to the number of spoVA 2mob copies on the genome, with a higher number of copies (up to three) correlating with increased heat resistance and reduced germination rate (18,19).…”

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Analysis of Germination Capacity and Germinant Receptor (Sub)clusters of Genome-Sequenced Bacillus cereus Environmental Isolates and Model Strains

Warda

Xiao

Boekhorst

et al. 2017

Appl Environ Microbiol

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Spore germination of 17 Bacillus cereus food isolates and reference strains was evaluated using flow cytometry analysis in combination with fluorescent staining at a single-spore level. This approach allowed for rapid collection of germination data under more than 20 conditions, including heat activation of spores, germination in complex media (brain heart infusion [BHI] and tryptone soy broth [TSB]), and exposure to saturating concentrations of single amino acids and the combination of alanine and inosine. Whole-genome sequence comparison revealed a total of 11 clusters of operons encoding germinant receptors (GRs): GerK, GerI, and GerL were present in all strains, whereas GerR, GerS, GerG, GerQ, GerX, GerF, GerW, and GerZ (sub)clusters showed a more diverse presence/absence in different strains. The spores of tested strains displayed high diversity with regard to their sensitivity and responsiveness to selected germinants and heat activation. The two laboratory strains, B. cereus ATCC 14579 and ATCC 10987, and 11 food isolates showed a good germination response under a range of conditions, whereas four other strains (B. cereus B4085, B4086, B4116, and B4153) belonging to phylogenetic group IIIA showed a very weak germination response even in BHI and TSB media. Germination responses could not be linked to specific (combinations of) GRs, but it was noted that the four group IIIA strains contained pseudogenes or variants of subunit C in their gerL cluster. Additionally, two of those strains (B4086 and B4153) carried pseudogenes in the gerK and gerR I (sub)clusters that possibly affected the functionality of these GRs.IMPORTANCE Germination of bacterial spores is a critical step before vegetative growth can resume. Food products may contain nutrient germinants that trigger germination and outgrowth of Bacillus species spores, possibly leading to food spoilage or foodborne illness. Prediction of spore germination behavior is, however, very challenging, especially for spores of natural isolates that tend to show more diverse germination responses than laboratory strains. The approach used has provided information on the genetic diversity in GRs and corresponding subclusters encoded by B. cereus strains, as well as their germination behavior and possible associations with GRs, and it provides a basis for further extension of knowledge on the role of GRs in B. cereus (group member) ecology and transmission to the host.

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Structural and Functional Analysis of the GerD Spore Germination Protein of Bacillus Species

Cited by 21 publications

References 45 publications

The Bacillus subtilis germinant receptor GerA triggers premature germination in response to morphological defects during sporulation

The Bacillus subtilis germinant receptor GerA triggers premature germination in response to morphological defects during sporulation

Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

Analysis of Germination Capacity and Germinant Receptor (Sub)clusters of Genome-Sequenced Bacillus cereus Environmental Isolates and Model Strains

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