Characterization of germination receptors of Bacillus cereus ATCC 14579

Abstract

Specific amino acids, purine ribonucleosides, or a combination of the two is required for efficient germination of endospores of Bacillus cereus ATCC 14579. A survey including 20 different amino acids showed that l-alanine, l-cysteine, l-threonine, and l-glutamine are capable of initiating the germination of endospores of B. cereus ATCC 14579. In addition, the purine ribonucleosides inosine and adenosine can trigger germination of the spores. Advanced annotation of the B. cereus ATCC 14579 genome revealed the presence of seven putative germination (ger) operons, termed gerG, gerI, gerK, gerL, gerQ, gerR, and gerS. To determine the role of the encoded putative receptors in nutrient-induced germination, disruption mutants were constructed by the insertion of pMUTIN4 into each of the seven operons. Four of the seven mutants were affected in the germination response to amino acids or purine ribonucleosides, whereas no phenotype could be attributed to the mutants with disrupted gerK, gerL, and gerS loci. The strain with a disrupted gerR operon was severely hampered in the ability to germinate: germination occurred in response to l-glutamine but not in the presence of any of the other amino acids tested. The gerG mutant showed significantly reduced l-glutamine-induced germination, which points to a role of this receptor in the l-glutamine germination signaling pathway. gerR, gerI, and gerQ mutants showed reduced germination rates in the presence of inosine, suggesting a role for these operons in ribonucleoside signaling. Efficient germination by the combination of l-glutamine and inosine was shown to involve the gerG and gerI operons, since the germination of mutants lacking either one of these receptors was significantly reduced. Germination triggered by the combination of l-phenylalanine and inosine was lost in the gerI mutant, indicating that both molecules are effective at the GerI receptor.

FIG. 1.

Germination survey of B. cereus ATCC 14579 wild-type spores and spores of the seven ger mutant strains with 20 amino acids. The percentage of germination was monitored after 60 min of incubation with the germinant molecules. The bars represent the responses of spores of the wild-type, gerR mutant, gerQ mutant, gerG mutant, and gerI mutant strains. The responses of the mutants with disrupted gerS, gerK, and gerL operons were similar to those of wild-type spores and were omitted for clarity. The amino acids alanine (A) and proline (not shown) were surveyed as single germinants, and the remaining amino acids were surveyed in groups in the following combinations: FTY (phenylalanine, tyrosine, and tryptophan), GVLI (glycine, valine, leucine, and isoleucine), CMST (cysteine, methionine, serine, and threonine), a group with lysine, arginine, and histidine (not shown), and DENQ (aspartate, glutamate, asparagine, and glutamine). The final concentration of each amino acid in these groups was 1 mM, except for tyrosine (0.5 mM). The addition of proline and the mixture of lysine, arginine, and histidine did not result in germination in any of the strains, and these results were omitted from the figure for clarity. Calcium DPA (CaDPA)-induced germination proceeds indirectly by activation of the cortex lytic enzymes, bypassing the germination receptors. Spores of all strains should have responded equally, and this was evaluated by assessing the germination response upon the addition of 50 mM calcium DPA. The results shown are the averages of duplicate experiments completed with two independent spore batches.

FIG. 2.

Effects of the amino acids alanine (1 mM), cysteine (1 mM), threonine (1 mM), and glutamine (1 mM) on the germination of spores of the wild-type strain and the gerR and gerG mutant strains. Germination was monitored as the fall in OD₆₀₀ over 60 min. ▾, wild-type strain with 1 mM cysteine; •, wild-type strain with 1 mM alanine; ▴, wild-type strain with 1 mM threonine; ▪, wild-type strain with 1 mM glutamine; ○, gerR strain with 1 mM alanine; □, gerG strain with 1 mM glutamine; ⋄, wild-type strain with no germinant. Germination data for gerR spore responses to cysteine and threonine were identical to those for the response to alanine and were omitted for clarity. Germination data for the gerR, gerQ, gerK, gerL, gerS, and gerI mutant strains upon the addition of l-glutamine were similar to those for the wild type and were omitted for clarity.

FIG. 3.

Effects of the purine ribonucleosides inosine and adenosine on the germination of spores of the wild-type strain and ger mutant strains. The bars represent the responses of spores of the wild-type, gerR mutant, gerQ mutant, and gerI mutant strains. The responses of the mutants with disrupted gerG, gerK, gerL, and gerS operons were similar to those of wild-type spores and were omitted for clarity.

FIG. 4.

Effect of 1 mM inosine on the germination of spores of the wild-type strain and the gerR, gerI, and gerQ mutants. •, wild-type strain; ▵, gerQ spores; ▿, gerI spores; ○, gerR spores; ⋄, wild-type strain with no germinant. Spores of these mutants showed reduced germination rates compared to that of the wild type, although the spores of these strains were able to complete germination within 60 min. The responses of gerG, gerK, gerL, and gerS spores were coincident with those of wild-type spores.

FIG. 5.

Germination survey of B. cereus wild-type spores and spores of the seven mutant strains with 20 amino acids, with inosine as a cogerminant. The concentrations of the amino acids used during this experiment were the same as those described in the legend to Fig. 1. The bars represent the responses of spores of the wild-type, gerR mutant, gerQ mutant, gerG mutant, and gerI mutant strains. The responses of mutant strains with disrupted gerK, gerL, and gerS operons were similar to those of wild-type spores and were omitted for clarity.

FIG. 6.

(A) Effect of 1 mM phenylalanine with 0.01 mM inosine as a cogerminant on the germination of spores of the wild-type strain (•), the gerR mutant (○), and the gerI mutant (▿). ⋄, wild-type strain without a germinant. Spores of the gerQ, gerG, gerK, gerL, and gerS mutant strains germinated like spores of the wild type (data not shown). (B) Effect of 1 mM glutamine with 0.01 mM inosine as a cogerminant on the germination of spores of the wild-type strain (•), gerR mutant spores (○), gerG mutant spores (□), and gerI mutant spores (▿). ⋄, wild-type strain without a germinant. Spores of strains with disrupted gerQ, gerK, gerL, and gerS loci responded similarly to spores of the wild-type strain (data not shown).

FIG. 7.

Unrooted phylogenetic tree based on the ger operons of B. cereus ATCC 14579 (GenBank accession number AE016877) (1), B. cereus ATCC 10987 (AE017194) (2), B. cereus 569 (AAD03541 to AAD03543 [gerI], AAK70461 to AAK70463 [gerL], and AAK63174 to AAK63176 [gerQ]) (3), and B. anthracis Sterne (AE017225) (4) and the plasmid-located gerX sequence derived from the genome sequence of B. anthracis A2012 (AE016879) (5). The tree is based on the amino acid sequence of the A cistron of each operon. The A cistrons of these strains clustered in eight distinct groups. If the operon annotation for the strain is distinct from the group name, the strain annotation is noted.