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. 2025 Oct 10;9:100485. doi: 10.1016/j.crmicr.2025.100485

Characterization of germinant receptors of P. thermoglucosidasius NCIMB 11955

Maika Salvador a, Matthew SH Lau b, Nigel P Minton b, Santiago Condón a, Elisa Gayán a,⁎
PMCID: PMC12554149  PMID: 41146727

Highlights

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    P. thermoglucosidasius NCIMB 11955 contains three germinant receptor orthologs.

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    GerK1 is essential for nutrient-induced germination.

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    GerK1 cooperates with GerX1 and/or GerK1 to fully induce germination in some sugars.

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    GerK1 is also key in colony formation but needs both GerK2/X1 for maximal recovery.

Keywords: Parageobacillus thermoglucosidasius, Spores, Germinant receptors, Germination, CRISPR/Cas9

Abstract

Germinant receptors (GRs) initiate spore germination in response to nutrient signals. While their roles are well studied in Bacillus spp., less is known in strict thermophiles such as (Para)Geobacillus spp. This study evaluated the roles of the GR operons identified in Parageobacillus thermoglucosidasius NCIMB 11955 (gerK1, gerK2, and gerX1) in germination and outgrowth. The GerK1 ortholog was essential for germination in TSB supplemented with yeast extract (TSBYE), D-fructose, and D-mannose; it cooperated with GerX1 for germination in maltose, sucrose, and raffinose; with GerK2 or GerX1 for germination in D-arabinose, D-glucose, and D-xylose; and with both for germination in lactose. GerK1 also dominated colony formation on TSA supplemented with yeast extract (TSAYE), but maximal recovery required GerX1 and GerK2, suggesting their involvement in spore outgrowth.

Graphical abstract

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1. Introduction

Under starvation, some bacteria initiate sporulation, producing metabolically dormant, highly resistant spores. When conditions improve, spores germinate and return to vegetative growth. In nature, germination is typically triggered by certain nutrients—mainly amino acids, nucleosides, and sugars—termed germinants. These are recognized by germinant receptors (GRs) in the inner membrane (Christie and Setlow, 2020). Each GR recognizes specific germinants, alone or combined, with overlapping specificities often requiring cooperation for maximal response (Atluri et al., 2006; Borch-Pedersen et al., 2016). Upon activation, GRs trigger Ca2+-dipicolinic acid (CaDPA) and ions release, core rehydration, cortex hydrolysis, and subsequent vegetative outgrowth (Christie and Setlow, 2020). Most spore-forming bacteria harbor multiple GR operons (Paredes-Sabja et al., 2011; Warda et al., 2017), typically expressed under the forespore-specific sigma factor σG (Wang et al., 2006; Steil et al., 2005). These operons are usually tricistronic, encoding subunits A, B, and C, though some species also have a D subunit that modulates germination (Paredes-Sabja et al., 2011; Gupta et al., 2015; Ramirez-Peralta et al., 2013). While subunit roles are not fully defined, evidence suggests the B subunit aids nutrient recognition (Gao et al., 2023; Artzi et al., 2021) and the A subunit participates in signal transduction (Amon et al., 2022).

The Parageobacillus and Geobacillus genera (collectively, (Para)Geobacillus) are spore-forming thermophiles growing over a broad temperature range (35–80 °C) (Studholme, 2015). Their highly heat-resistant spores cause spoilage in shelf-stable thermally processed foods (André et al., 2013), while their thermophilic traits enable applications in food safety (e.g., antibiotic residue detection kits), bioremediation, biofuel production, and thermostable enzyme synthesis (Li et al., 2021; Salvador et al., 2025b). Among (Para)Geobacillus, the type Parageobacillus thermoglucosidasius strains (DSM 2542 and NCIMB 11955) have been engineered for biofuels (Sheng et al., 2023), riboflavin (Wang et al., 2024), and lactate (Liu et al., 2024) production, among others.

Understanding GR profiles and functions in (Para)Geobacillus is essential for controlling food spoilage and optimizing spore-based biotechnological uses. Although we recently delineated GR operon diversity and characterized germination in representative strains (Salvador et al., 2025c; Salvador et al., 2025a), the nutrient-specific roles of individual GR types remain largely unknown. Here, we investigated the role of each GR operon in P. thermoglucosidasius NCIMB 11955, using a restriction-modification-deficient derivative (Lau, 2018) to construct mutants lacking a single GR operon or retaining only one, and tested their responses to diverse nutrient germinants.

2. Material and methods

2.1. Genome mining

For identification of GRs, the genome of P. thermoglucosidasius NCIMB 11955 (NZ_CP016622) was re-annotated and orthologous groups were predicted using GR sequences of reference strains (Bacillus subtilis 168, B. anthracis Ames Ancestor, and Priestia megaterium QM B1551) as described by Salvador et al. (2025c).

Promoter regions upstream of selected genes were aligned, analyzed, and visualized using Clustal-Omega (https://ngphylogeny.fr/workflows/alacarte) (Sievers et al., 2011; Capella-Gutiérrez et al., 2009; Lemoine et al., 2019; Yachdav et al., 2016) to identify putative sporulation sigma factor binding sites, based on consensus sequences from B. subtilis (DBTBS, a database of transcriptional regulation in B. subtilis; https://dbtbs.hgc.jp) (Makita et al., 2004) and B. licheniformis ATCC 14580 (Borch-Pedersen et al., 2016). Schematic representations of the predicted GR operon structures were created using SnapGene® software v.7.0.1 (from Dotmatics; available at https://snapgene.com).

2.2. Strain construction and growth conditions

Bacterial strains (Table S1) were stored at −80 °C in 25 % (v/v) glycerol (Panreac, Barcelona, Spain). Escherichia coli Top 10 (Invitrogen Ltd., Waltham, MA, USA) was used for cloning and grown in 5 mL of LB broth (Merck Millipore, Rahway, NJ, USA) or on LB agar (15 g/L; No. 1 bacteriological agar, Oxoid, Basingstoke, UK) with kanamycin (Km; 50 µg/mL; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C overnight (O/N) (Lau et al., 2021). P. thermoglucosidasius NCIMB 11955 (TMO Renewables Ltd., London, UK) and ML015, a derivative lacking three restriction–modification systems for improved transformation (Lau, 2018), were grown in 10 mL of 2SPYNG broth (soy peptone (16 g/L, Solabia, Cedex, France), yeast extract (10 g/L, Oxoid), and NaCl (5 g/L, Panreac) at pH 7.0) or on TSA (Oxoid) plates at 52 °C O/N.

Markerless GR operon deletions were generated by CRISPR/Cas9 using pMTL675555 as the backbone vector (Lau et al., 2021; https://www.plasmidvectors.com) and primers listed in Table S2. Plasmids were transformed into P. thermoglucosidasius by electroporation (Cripps et al., 2009). Constructs were confirmed by PCR with primers flanking the engineered region (Table S2) and verified by sequencing (Source Bioscience UK Limited, Nottingham, UK).

2.3. Sporulation

For spore production, single colonies were grown in 20 mL of 2TY (Sigma-Aldrich) at 55 °C O/N. Then, 200 µL were transferred to 50 mL of TYE sporulation medium (0.4 % (w/v) tryptone (VWR International Chemicals, Radnor, PA, USA), 0.4 % (w/v) yeast extract, 1.13 mM CaCl2·2H₂O (VWR International Chemicals), 0.033 mM MnSO4·H₂O (CARLO ERBA Reagents GmbH, Cornaredo, Milan, Italy), 1 mM MgSO4·7H₂O (Panreac), 0.04 mM FeSO4·7H₂O (Sigma-Aldrich), and 80 mM HEPES buffer pH 7.0 (Sigma-Aldrich)) and incubated at 55 °C for 4 days (Salvador et al., 2025a). Spores were harvested by centrifugation followed by water washes with an intermediate ethanol treatment (Salvador et al., 2025a), purified by Nycodenz® buoyant density centrifugation (Ghosh and Setlow, 2009; Freire et al., 2023), and stored in distilled water at −20 °C. Purity (99 % phase-bright) was confirmed by phase-contrast microscopy (Nikon Eclipse E400, Tokyo, Japan). Biological variability was assessed using three independent spore batches.

2.4. Germination assay and colony-forming efficiency

Spore germination kinetics were monitored as OD₆₀₀ decline, reflecting CaDPA release and spore hydration. Suspensions were adjusted to OD₆₀₀ 0.4–0.6 in germination medium. Nutrient germinants—including l-amino acids, nucleosides, casamino acids, and TSB supplemented with yeast extract and chloramphenicol—and the non-nutrient germinant CaDPA were tested at concentrations sufficient for maximal germination (Salvador et al., 2025a). OD₆₀₀ was recorded every 3 min for 4 h at 55 °C (CLARIOstar Plus, BMG, Ortenberg, Germany). Germination efficiency was calculated as the decrease in the OD600 relative to the initial value (ODt/ODi × 100, where ODt and ODi are final and initial OD₆₀₀ values, respectively), which correlated with the proportion of phase-dark spores by microscopy using the following thresholds: ≤ 15 % OD₆₀₀ decrease corresponded to ≤ 5 % germinated spores, and > 45 % to > 90 % germinated.

Colony-forming efficiency was calculated as NC/NT × 100, where NT is the n° spores/mL determined microscopically, and NC is the CFU/mL obtained on TSA supplemented with 0.6 % (w/v) yeast extract (TSAYE; 55 °C, 48 h).

2.5. Statistical analysis

Statistical analyses were performed in GraphPad Prism 8.4.2 (GraphPad Software, San Diego, CA, USA) using unpaired t-tests and one-way ANOVA with Tukey’s post hoc test. Significance was set at p ≤ 0.05. Data are means ± standard deviation from ≥ 3 biological replicates.

3. Results and discussion

3.1. Characterization of GRs in silico

As the main confirmed differences between P. thermoglucosidasius NCIMB 11955 and DSM 2542 consist of 11 single-nucleotide polymorphisms and two indels affecting genes unrelated to germination (Sheng et al., 2016), it is not surprising that, as in P. thermoglucosidasius DSM 2542 (Salvador et al., 2025c), strain NCIMB 11955 also harbors three distinct GRs: two in tricistronic operons (gerK1 and gerX1) and one (gerK2) in a tetracistronic operon (Fig. 1). The gerK2 operon encoded a hypothetical protein with low similarity to YfkS—the D subunit of the Yfk receptor in B. subtilis 168—sharing 95 % query coverage but only 25.0 % identity. Another hypothetical protein, with 93 % coverage and 36.6 % identity to GerUD from Priestia megaterium QM B1551, was found adjacent to gerKB of gerK1, although in a separate transcriptional unit. Promoter regions upstream of the GR operons were predicted using the σG consensus sequence from B. subtilis (Makita et al., 2004) and B. licheniformis ATCC 14580 (Borch-Pedersen et al., 2016) (Fig. 1).

Fig. 1.

Fig 1

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Organization of the P. thermoglucosidasius NCIMB 11955 gerA-family operons: gerK1 (BCV53_13,760, BCV53_13,765, BCV53_13,770; 3958 bp), gerX1 (BCV53_13,830, BCV53_13,835, BCV53_13,840; 3758 bp), and gerK2 (BCV53_15,720, BCV53_15,725, BCV53_15,730, BCV53_15,735; 4168 bp). Putative promoter regions are indicated with arrows, based on the σG consensus from B. subtilis (Makita et al., 2004) and B. licheniformis ATCC 14580 (Borch-Pedersen et al., 2016).

To assess each GR’s role and cooperativity in germination and colony-forming efficiency, we deleted operons individually. In P. thermoglucosidasius NCIMB 11955, gerK1 and gerK2 were deleted, but gerX1 deletion failed. Using the higher-efficiency strain ML015 (Lau, 2018), we generated ΔgerK1, ΔgerK2, and ΔgerX1 single mutants (Fig. S1). Sequential deletions yielded gerK1+, gerK2+, and gerX1+ strains, each retaining only one functional GR. Triple-deletion attempts failed, as no colonies formed after transformation.

3.2. Germination efficiency

Germination of parental ML015 spores and GR-deficient mutants was assessed in liquid media containing individual nutrients or a complex mixture (TSBYE), based on OD₆₀₀ decrease over 4 h (Table 1). As expected from its genetic similarity to P. thermoglucosidasius DSM 2542 (Salvador et al., 2025c), the parental strain showed no germination (OD600 decrease ≤ 15.0 %) in l-amino acids or nucleosides but responded to TSBYE and various sugars. In GR-deficient mutants, deletion of gerK1—either alone or in combination with deletion of gerK2 (gerX1+) or gerX1 (gerK2+)—abolished germination in all nutrients. Retention of only gerK1 (gerK1+) allowed for germination similar to that of the parental strain in TSBYE, d-fructose, and d-mannose, but reduced germination in other sugars, suggesting that GerK1 cooperates with additional GRs. In d-arabinose, d-glucose, and d-xylose, gerK1+ spores, but not ΔgerX1 or ΔgerK2 spores, germinated less efficiently than the parental strain, implying that GerK1 requires support from GerK2 or GerX1 for full activity with these monosaccharides. In maltose, sucrose, and raffinose, ΔgerX1 spores also showed reduced germination compared to the parental strain, indicating cooperation between GerK1 and GerX1. For lactose, gerK1+, ΔgerX1, and ΔgerK2 spores all showed similarly reduced germination (p > 0.05) relative to the parental strain, suggesting that all three GRs are required for full induction by lactose. Collectively, these results indicate that GerK1 is essential for nutrient-induced germination, but needs cooperation with other GRs for efficient activation by specific sugars. The conservation of the gerK1 operon across all (Para)Geobacillus spp. strains (Salvador et al., 2025c) further suggests that this GR plays a central role in spore revival in these genera.

Table 1.

Germination response of the parental ML015 strain and the indicated mutants to various germinants. Germination was evaluated based on the OD600nm decrease after 4 h incubation at 55 °C. The lower and upper detection limits for OD₆₀₀ decrease were defined as ≤ 15.0 % (≤ 5 % germination) and > 45.0 % (> 90 % germination), respectively. Data in brackets represent standard deviations of the means from three biological replicates.

Germinants ML015 ΔgerK1 ΔgerX1 ΔgerK2 gerK1+ gerX1+ gerK2+
TSBYE > 45.0 ≤ 15.0 > 45.0 > 45.0 > 45.0 ≤ 15.0 ≤ 15.0
l-amino acids (0.5 – 100 mM)* ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0
Casamino acids (2 %) ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0
Adenosine (7.5 mM) ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0
Inosine (25 mM) ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0 ≤ 15.0
d-Arabinose (100 mM) > 45.0 ≤ 15.0 > 45.0 > 45.0 29.0 (3.3) ≤ 15.0 ≤ 15.0
d-Fructose (100 mM) > 45.0 ≤ 15.0 > 45.0 > 45.0 > 45.0 ≤ 15.0 ≤ 15.0
d-Glucose (100 mM) > 45.0 ≤ 15.0 44.5 (0.8)a > 45.0 25.4 (3.0)b ≤ 15.0 ≤ 15.0
d-Mannose (100 mM) > 45.0 ≤ 15.0 > 45.0 > 45.0 > 45.0 ≤ 15.0 ≤ 15.0
d-Xylose (100 mM) > 45.0 ≤ 15.0 > 45.0 > 45.0 32.3 (3.3) ≤ 15.0 ≤ 15.0
Lactose (100 mM) 41.5 (1.1)a ≤ 15.0 29.6 (1.1)b 25.5 (1.7)b 27.6 (3.4)b ≤ 15.0 ≤ 15.0
Maltose (100 mM) > 45.0 ≤ 15.0 35.0 (1.0)a > 45.0 33.6 (1.0)a ≤ 15.0 ≤ 15.0
Sucrose (100 mM) > 45.0 ≤ 15.0 33.1 (1.3)a > 45.0 29.2 (1.0)a ≤ 15.0 ≤ 15.0
Raffinose (100 mM) > 45.0 ≤ 15.0 36.0 (1.6)a > 45.0 23.4 (2.8)b ≤ 15.0 ≤ 15.0
CaDPA (60 mM) > 45.0 > 45.0 > 45.0 > 45.0 > 45.0 > 45.0 > 45.0
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⁎

Each l-amino acid was tested at 100 mM, except for l-phenylalanine (30 mM), l-threonine (0.5 mM), l-aspartic acid (10 mM), and l-glutamic acid (25 mM).

a, b Letters allow data to be statistically compared. In each germinant, there are significant differences (p ≤ 0.05) among strains when they do not share the same letter.

Previous studies have reported that gerK homologs play a critical role in germination in both nutrient-rich media and sugars, either alone or in the presence of co-germinants, in spore-forming bacteria. However, as in the case of P. thermoglucosidasius, GerK receptors often function cooperatively with other GR types. In B. subtilis PS832, gerK contributed to glucose recognition in combination with l-asparagine, d-glucose, d-fructose, and KCl (AGFK), but required the presence of gerB for efficient germination (Atluri et al., 2006). In B. licheniformis ATCC 14580, the absence of gerK inhibited the weak response to d-glucose, but full induction required the presence of gerAB and gerAC (Borch-Pedersen et al., 2016). In Priestia megaterium PV361 (a QM B1551 derivative lacking the plasmid-borne gerU), the chromosomal gerK1 ortholog was essential for responding to beef extract and LB; however, its absence was partially compensated by deleting the other functional gerK2 operon (Gupta et al., 2013).

Positive cooperativity among GRs, combined with differences in affinity and specificity, likely broadens spore germinant recognition, enabling revival in diverse niches (Carr et al., 2010; Gupta et al., 2013). The molecular basis remains unclear but may involve regulatory control of GR locus expression and/or modulation of germinant access or integrated receptor function via structural clustering of GRs with signal transducers in the germinosome (Li et al., 2014; Moir and Cooper, 2015; Gupta et al., 2013).

3.3. Colony-forming efficiency

The colony-forming efficiency of all strains was evaluated on TSAYE (48 h, 55 °C), reflecting both spore germination and subsequent outgrowth in the complex medium (Fig. 2). The parental strain ML015 formed colonies at 74.3 %, similar to wild-type NCIMB 11955 (data not shown). This efficiency is lower than that reported in mesophilic Bacillus spp. (> 95 %), such as B. subtilis, B. anthracis, and Priestia megaterium (Gupta et al., 2013; Carr et al., 2010; Ghosh and Setlow, 2009), but relatively high compared to other (Para)Geobacillus spp., some of which show ≤ 10 % (Huesca-Espitia et al., 2016; Salvador et al., 2025c).

Fig. 2.

Fig 2

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Colony-forming efficiency (expressed as the percentage of total spores, determined microscopically, that form colonies on TSAYE plates (55 °C, 48 h)) of the parental ML015 strain and the indicated mutants. Data represent mean values and standard deviations from three biological replicates. Letters above the bars allow data to be statistically compared. There are significant differences (p ≤ 0.05) among strains when they do not share the same letter.

All GR-deficient spores exhibited significantly reduced (p ≤ 0.05) colony-forming efficiency compared to the parental strain. Deletion of gerK1, which also inhibited short-term (4 h) germination in the same liquid medium, reduced efficiency 19.1-fold. Mutants lacking gerK2, gerX1, or both (gerK1+) showed smaller reductions (1.3–1.7-fold). The presence of only one of the minor GR operons, gerK2+ or gerX1+, decreased (p ≤ 0.05) the colony-forming efficiency compared to the gerK1+ spores by 37.9-fold and 161.9-fold, respectively (i.e., 50.8- and 217.2-fold reductions compared to the parental strain, respectively). Although a GR-null mutant could not be obtained in P. thermoglucosidasius, spores of Bacillus spp. lacking all GR operons show extremely low colony-forming efficiency (0.03–0.001 %) (Gupta et al., 2013; Paidhungat and Setlow, 2000; Borch-Pedersen et al., 2017), far below that of the most impaired mutant here (gerX1+, 0.34 %). These results indicate that while GerK1 is the primary receptor for revival on TSAYE, all three GRs are required for maximal recovery.

All predicted GR-encoding operons in P. thermoglucosidasius contributed to maximal colony formation—especially gerK1, followed by gerK2 and gerX1—in contrast to other spore-formers. In B. subtilis PS832, deletion of gerA moderately reduced colony formation in LB (2.4-fold), while loss of gerK, gerB, or both had negligible effects (Paidhungat and Setlow, 2000). In Priestia megaterium QM B1551, loss of the plasmid-borne gerU did not affect LB colony counts, likely due to the presence of gerK1. However, simultaneous deletion of gerU and gerK1 reduced colony formation 100.0-fold, although additional inactivation of gerK2 partially restored it to 60 % (Christie and Lowe, 2007; Gupta et al., 2013). In Clostridium perfringens SM101, disruption of the gerKA ortholog, but not gerAA, led to a 52.5-fold decrease in colony formation (Paredes-Sabja et al., 2008).

Notably, ΔgerK2, ΔgerX1, and gerK1+ spores germinated proficiently in TSBYE but showed reduced colony-forming efficiency (Fig. 2), suggesting that GerK2 and GerX1 support maximal recovery during outgrowth rather than germination. Similarly, in C. perfringens SM101, the gerKA mutant showed a smaller decrease in germination than in colony formation, although gerKA also contributed to CaDPA-induced germination (Paredes-Sabja et al., 2008). In contrast, all P. thermoglucosidasius mutants and the parental strain responded equally to CaDPA (Table 1), evidencing that this pathway is GR-independent, as previously reported for Bacillus spp. (Løvdal et al., 2012; Paidhungat et al., 2001). Thus, the role of GerK in C. perfringens outgrowth may differ from that of GerX1 and GerK2 in P. thermoglucosidasius, likely independent of CLE activity.

4. Conclusions

This is the first study to examine GR function in thermophilic spores of the (Para)Geobacillus genera, using a genetically tractable derivative of the type strain P. thermoglucosidasius as a model. Among the three predicted GR operons (gerK1, gerK2, and gerX1), GerK1 was the primary receptor for germination in complex medium and with d-fructose or d-mannose. Its full activity required cooperation with GerK2, GerX1, or both for efficient germination with other sugars. While GerK2 and GerX1 played minor roles in short-term germination in complex medium, both were essential for maximal colony-forming efficiency, suggesting an additional role during outgrowth.

CRediT authorship contribution statement

Maika Salvador: Conceptualization, Formal analysis, Investigation, Methodology, Writing- original draft, Writing- review & editing. Matthew Lau: Methodology, Writing- review & editing. Nigel Minton: Writing- review & editing. Santiago Condón: Conceptualization, Supervision, Funding Acquisition, Writing – review & editing. Elisa Gayán: Conceptualization, Methodology, Funding Acquisition, Project administration, Resources, Supervision, Writing- original draft. Writing – review & editing.

Data availability

Data will be made available on request.

Funding

This study was supported by grants (TED2021–129779B-I00 and PID2023–148505OB-I00) from MCIN/AEI/10.13039/501100011033, and a Ph.D. scholarship from the Government of Aragón (to M.S.).

Acknowledgments

The authors would also like to acknowledge “Servicio General de Apoyo a la Investigación-SAI” (University of Zaragoza) for developing the automatic colony counting system, and ChatGPT for language support. We would like to express our sincere gratitude to Professor Santiago Condón Usón, whose insightful discussions and contributions greatly enriched this work. Professor Santiago Condón Usón passed away during the preparation of this manuscript. We honor his memory and scientific legacy.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.crmicr.2025.100485.

Appendix. Supplementary materials

mmc1.docx (815.2KB, docx)

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Supplementary Materials

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