Investigating the Functional Hierarchy of Bacillus megaterium PV361 Spore Germinant Receptors

Abstract

Spores of Bacillus megaterium QM B1551 germinate rapidly when exposed to a number of single-trigger germinant compounds, including glucose, proline, leucine, and certain inorganic salts. However, spores of strain PV361, a plasmidless QM B1551 derivative that lacks the GerU germinant receptor (GR) responsible for mediating germination in response to single-trigger compounds, can germinate efficiently when incubated in nutritionally rich media, presumably via activation of additional germinant receptors. In this work, we have identified five chromosomally encoded GRs and attempted to characterize, by mutational analysis, germinant recognition profiles associated with the respective receptors in strain PV361. Of strains engineered with single GR insertion-deletions, only GerK-null spores displayed significant defective germination phenotypes when incubated in 5% (wt/ vol) beef extract or plated on rich solid medium. Cumulative decreases in viability were observed in GerK-null spores that also lacked GerA or GerA 2 , indicating that these GRs, which exerted little effect on spore germination when disrupted individually, have a degree of functionality. Unexpectedly, an efficient germination response to combinations of germinants was restored in GerA ؉ spores, which lack all other functional GRs, providing evidence for negative cooperativity between some GRs within the spore. Tetrazolium-based germinative assays conducted with purified spores indicated that these newly characterized B. megaterium GRs are cognate for a wide and chemically diverse range of germinant molecules, but unlike GerU, can only be induced to trigger germination when stimulated by at least two different germinants.

Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination

“…B. megaterium spore viability was determined at 30°C to minimize mucoid colony formation, which interferes with the formation of isolated colonies. The viability of treated and purified FB73 and GC614 spores was not measured, as these spores germinate extremely poorly with nutrient germinants (15,18).…”

“…The GR-dependent germinants used were as follows: B. subtilis spores, 10 mM L-valine or a mixture of 10 mM L-asparagine, 10 mM D-glucose, 10 mM D-fructose, and 10 mM KCl (AGFK); B. megaterium spores, 10 mM D-glucose or 50 mM KBr. Tb-DPA fluorescence was read every 5 min for 30 min to 2 h. Untreated spores were heat activated prior to germination as follows: 30 min or 2 h at 75°C for B. subtilis spores germinating with L-valine or AGFK, respectively, and 30 min at 60°C for B. megaterium spores (18,23). After heat activation, spores were cooled on ice for at least 15 min prior to germination experiments.…”

Analysis of Metabolism in Dormant Spores of Bacillus Species by 31 P Nuclear Magnetic Resonance Analysis of Low-Molecular-Weight Compounds

“…The B. megaterium strains used were QM B1551 (wild type) (originally obtained from H. S. Levinson) and two isogenic derivatives, GC614 (13), lacking genes encoding all functional GRs, and PS4241 (15), lacking the genes encoding the two redundant cortex-lytic enzymes (CLEs), CwlJ and SleB, that degrade spore cortex peptidoglycan during germination. The B. subtilis strains used were PS533 (wild type) (18), a prototrophic laboratory 168 strain that also carries plasmid pUB110 expressing kanamycin resistance, and an isogenic derivative, FB73 (14), that lacks plasmid pUB110 and genes for all functional GRs.…”

“…However, a potentially simpler way to deal with this problem is to use spores that do not respond to external nutrient germinants because they lack the spore proteins that recognize and trigger germination in response to nutrient germinants (2). These proteins are termed germinant receptors (GRs), and molecular genetic manipulation has generated strains of B. megaterium and B. subtilis that lack all functional GRs (13,14).…”

Germination of Spores of Bacillus Species: What We Know and Do Not Know

“…The GRs are clearly of crucial importance to nutrient germination, as spores of several Bacillus species that have been engineered to lack functional GRs exhibit extremely low levels of germination-as low as 0.1% per day on nutrient media (46,47). The precise mechanism of this spontaneous GR-independent germination is not clear, but it requires at least one CLE, since Bacillus spores that lack CwlJ and SleB exhibit Ͻ0.001% of the colony-forming ability of wild-type spores (48).…”