Germinant Receptor Protein Research Articles

Genomic versus Plasmid-Borne Expression of Germinant Receptor Proteins in Bacillus cereus Strain 14579

Germinant receptors (GRs) are proteins in the spore-forming bacteria of Bacillus species that are crucial in triggering spore germination by sensing nutrients in the spores’ environment. In the Gram-positive bacterium Bacillus cereus strain ATCC 14579, the GerR GR initiates germination with L-alanine. While we have expressed GerR subunits fused to reporter proteins from genes under control of their native promoter on plasmids in this B. cereus strain, here we sought increased flexibility in this work by studying genome integration and plasmid-borne inducible high level (over) expression. However, construction of chromosomal integrants to visualize and localize the GerR B subunit fused to fluorescent reporter protein SGFP2 was not successful in this B. cereus strain using constructs with either shorter (~600 bp) or longer (~1200 bp) regions of homology to the gerR operon. This failure was in contrast to successful IPTG-inducible expression of GerRB-SGFP2 from plasmid pDG148 in vegetative cells and dormant spores, as fluorescent GerRB-SGFP2 foci were present in vegetative cells and the protein was detected by Western blot analysis. In dormant spores, the fluorescence intensity with IPTG-inducible expression from pDG148-gerRB-SGFP2 was significantly higher than in wild type spores. However, the full length GerRB-SGFP2 protein was not detected in spores using Western blots. Clearly, there are still challenges in the construction of B. cereus strains harboring fluorescent reporter proteins in which tagged proteins are encoded by genes incorporated in the chromosome or on extrachromosomal expression plasmids.

The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II

Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca2+-DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II.

Analysis of the Germination of Individual Clostridium sporogenes Spores with and without Germinant Receptors and Cortex-Lytic Enzymes.

The Gram-positive spore-forming anaerobe Clostridium sporogenes is a significant cause of food spoilage, and it is also used as a surrogate for C. botulinum spores for testing the efficacy of commercial sterilization. C. sporogenes spores have also been proposed as a vector to deliver drugs to tumor cells for cancer treatments. Such an application of C. sporogenes spores requires their germination and return to life. In this study, Raman spectroscopy and differential interference contrast (DIC) microscopy were used to analyze the germination kinetics of multiple individual C. sporogenes wild-type and germination mutant spores. Most individual C. sporogenes spores germinated with L-alanine began slow leakage of ∼5% of their large Ca-dipicolinic acid (CaDPA) depot at T1, all transitioned to rapid CaDPA release at Tlag1, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlys. T1, Tlag1, Trelease, and Tlys times for individual spores were heterogeneous, but ΔTrelease (Trelease – Tlag1) periods were relatively constant. However, variability in T1 (or Tlag1) times appeared to be the major reason for the heterogeneity between individual spores in their germination times. After Trelease, some spores also displayed another lag in rate of change in DIC image intensity before the start of a second obvious DIC image intensity decline of 25–30% at Tlag2 prior to Tlys. This has not been seen with spores of other species. Almost all C. sporogenes spores lacking the cortex-lytic enzyme (CLE) CwlJ spores exhibited a Tlag2 in L-alanine germination. Sublethal heat treatment potentiated C. sporogenes spore germination with L-alanine, primarily by shortening T1 times. Spores without the CLEs SleB or CwlJ exhibited greatly slowed germination with L-alanine, but spores lacking all germinant receptor proteins did not germinate with L-alanine. The absence of these various germination proteins also decreased but did not abolish germination with the non-GR-dependent germinants dodecylamine and CaDPA, but spores without CwlJ exhibited no germination with CaDPA. Finally, C. sporogenes spores displayed commitment in germination, but memory in GR-dependent germination was small, and less than the memory in Bacillus spore germination.

Characterization of germinants and their receptors for spores of non-food-borne Clostridium perfringens strain F4969.

Clostridium perfringens type A can cause both food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases. Our previous study reported that a mixture of l-asparagine and KCl (AK)-germinated spores of FP and NFB isolates well, but KCl and, to a lesser extent, l-asparagine induced spore germination only in FP isolates. We now report that the germination response of FP and NFB spores differsignificantly in several defined germinants and rich media. Spores of NFB strain F4969 gerAA, gerKA-KC or gerKC mutants lacking specific germinant receptor proteins germinated more slowly than wild-type spores with rich media, did not germinate with AK and germinated poorly compared to wild-type spores with l-cysteine. The germination defects in the gerKA-KC spores were largely due to loss of GerKC as (i) gerKA spores germinated significantly with all tested germinants, while gerKC spores exhibited poor or no germination; and (ii) germination defects in gerKC spores were largely restored by expressing the wild-type gerKA-KC operon in trans. We also found that gerKA-KC, gerAA and gerKC spores, but not gerKA spores, released dipicolinic acid at a slower rate than wild-type spores with AK. The colony-forming efficiency of F4969 gerKC spores was also ~35-fold lower than that of wild-type spores, while gerAA and wild-type spores had similar viability. Collectively, these results suggest that the GerAA and GerKC proteins play roles in normal germination of C. perfringens NFB isolates and that GerKC, but not GerAA, is important in these spores' apparent viability.

Levels of germination proteins in Bacillus subtilis dormant, superdormant, and germinating spores.

Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.

The Clostridium perfringens Germinant Receptor Protein GerKC Is Located in the Spore Inner Membrane and Is Crucial for Spore Germination

The Gram-positive, anaerobic, spore-forming bacterium Clostridium perfringens causes a variety of diseases in both humans and animals, and spore germination is thought to be the first stage of C. perfringens infection. Previous studies have indicated that the germinant receptor (GR) proteins encoded by the bicistronic gerKA-gerKC operon as well as the proteins encoded by the gerKB and gerAA genes are required for normal germination of C. perfringens spores. We now report the individual role of these GR proteins by analyzing the germination of strains carrying mutations in gerKA, gerKC, or both gerKB and gerAA. Western blot analysis was also used to determine the location and numbers of GerKC proteins in spores. Conclusions from this work include the following: (i) gerKC mutant spores germinate extremely poorly with KCl, l-asparagine, a mixture of asparagine and KCl, or NaPi; (ii) gerKC spores germinate significantly more slowly than wild-type and other GR mutant spores with a 1:1 chelate of Ca(2+) and dipicolinic acid and very slightly more slowly with dodecylamine; (iii) the germination defects in gerKC spores are largely restored by expressing the wild-type gerKA-gerKC operon in trans; (iv) GerKC is required for the spores' viability, almost certainly because of the gerKC spores' poor germination; and (v) GerKC is located in the spores' inner membrane, with ∼250 molecules/spore. Collectively, these results indicate that GerKC is the main GR protein required for nutrient and nonnutrient germination of spores of C. perfringens food-poisoning isolates.

Host serum factor triggers germination of Clostridium perfringens spores lacking the cortex hydrolysis machinery

Clostridium perfringens type A is the causative agent of a variety of histotoxic and enteric diseases. The ability of C. perfringens spores to germinate in vivo might be due to the presence of nutrient germinants in the host tissue and blood. In the current study, we investigated the ability of spores of C. perfringens wild-type and mutation strains to germinate in blood. Results indicate that spores of all three surveyed C. perfringens wild-type isolates germinated better in blood than in brain heart infusion (BHI) broth. However, as expected, spores lacking germinant receptor (GR) protein GerAA or GerKB germinated like wild-type spores in BHI broth and blood. Strikingly, while spores lacking GR proteins GerKA and GerKC showed significantly decreased germination in BHI broth, these spores germinated well in blood, suggesting that blood factor(s) can trigger spore germination through a GR-independent pathway. Using C. perfringens spores lacking cortex lytic enzymes (ΔcspB or ΔsleC ΔsleM), we were able to identify a host serum germination factor with peptidoglycan hydrolysing activity that (i) restored the colony-forming efficiencies of ΔcspB and ΔsleC ΔsleM spores up to ~5-20% of that of total colony-forming spores; (ii) increased the number of c.f.u. of decoated ΔcspB and ΔsleC ΔsleM spores to ~99% of that of colony-forming spores; (iii) and finally lost enzymic activity after heat inactivation, consistent with serum germination factor being an enzyme. Further characterization demonstrated that serum germination factor is very likely lysozyme, which can form a stable high molecular mass complex of ~120 kDa in serum. In conclusion, the current study indicates that a host serum germination factor with peptidoglycan hydrolysing activity is capable of triggering germination of C. perfringens spores by directly degrading the spore peptidoglycan cortex. Collectively, this study contributes to our understanding of the mechanism of in vivo germination of spores of C. perfringens.

Amino Acid Residues in the GerAB Protein Important in the Function and Assembly of the Alanine Spore Germination Receptor of Bacillus subtilis 168

The paradigm gerA operon is required for endospore germination in response to c-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006 León, Spain-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-amino-acid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore.

Localization of the GerD spore germination protein in the Bacillus subtilis spore

The GerD protein of Bacillus subtilis is required for efficient spore germination in l-alanine, and for germination in the alternative germinant combination of amino acids plus sugars. Only germination via nutrient receptors is affected in the mutant. The GerD protein is predicted to be a lipoprotein that is produced in the forespore compartment of the sporulating cell. Using antibody raised against the GerD protein, Western blots of proteins from spore fractions revealed that, as might be expected, the protein was detected in the inner membrane of spores, but it was also present at a high level in spore integuments (comprising coat, cortex and germ cell wall layers), and to some extent in the soluble fraction. It is likely that the GerD protein in the outer layers of dormant spores is located in the germ cell wall, as it was detected in coat-defective spores, and in the cell wall fraction of cells that were outgrowing from spores. Which of the multiple locations of GerD is important for its function is not known, but the inner membrane association would be appropriate for any interaction with germinant receptor proteins or SleB cortex lytic enzyme. Substitution of alanine for cysteine in the conserved cleavage site of the predicted prelipoprotein signal sequence of GerD resulted in mutant spores that lacked the GerD protein entirely.

Analysis of interactions between nutrient germinant receptors and SpoVA proteins ofBacillus subtilisspores

Yeast two-hybrid and Far Western analyses were used to detect interactions between Bacillus subtilis spores' nutrient germinant receptor proteins and proteins encoded by the spoVA operon, all of which are involved in spore germination and located in the spores' inner membrane. These analyses indicated that two subunits of the GerA nutrient germinant receptor interact, consistent with previous genetic data, and that some GerA proteins interact with SpoVAD and some with SpoVAE. SpoVA proteins appear to be involved in the release of the spore's dipicolinic acid during spore germination, an event triggered by the binding of nutrient germinants to their receptors. Consequently, these new findings suggest that nutrient germinant receptors physically contact SpoVA proteins, and presumably this is a route for signal transduction during spore germination.

Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores.

Dormant Bacillus subtilis spores germinate in response to specific nutrients called germinants, which are recognized by multisubunit receptor complexes encoded by members of the gerA family of operons, of which the gerB operon is a member. The germinant receptors are expected to be membrane associated, but there is some debate about whether they are located in the inner or outer spore membrane. In this study we have used Western blot analysis to determine the precise location of GerBA, a gerB-encoded receptor protein, in various spore fractions. GerBA was not extracted from spores by a decoating treatment that removes the coat and outer membrane but was present in lysates from decoated spores and in the insoluble fraction (termed P100) from such lysates that contained inner-membrane vesicles. GerBA was also solubilized from the P100 fraction with detergent but not with high salt. These findings suggest that GerBA is an integral membrane protein located in the spore's inner membrane. Consistent with this idea, GerBA was present in the cell membrane of the outgrowing spore, a membrane that is derived from the dormant spore's inner membrane. Based on these observations we propose that GerBA and probably the entire GerB germinant receptor are located in the inner membrane of the dormant spore. We also estimated that there are only 24 to 40 molecules of GerBA per spore, a number that is consistent with the previously reported low level of gerB operon expression and with the putative receptor function of the proteins encoded by the gerB operon.