GerN, an antiporter homologue important in germination of Bacillus cereus endospores

Abstract

A homologue of the grmA spore germination gene of Bacillus megaterium and of a NaH-antiporter gene (napA) of Enterococcus hirae has been identified in Bacillus cereus 569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this B. cereus gene, named gerN, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to L-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 microM), inosine germination was completely blocked in the mutant, while the rate of germination in 50 microM L-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of L-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations.

FIG. 1

Multiple sequence alignment of the GerN protein against its homologues Gba_1701 of B. anthracis, GrmA of B. megaterium, NapA of E. hirae, locus CAA51756 of L. lactis, the N-terminal domains of KefB and KefC of E. coli, and the N-terminal domain of YjbQ of B. subtilis. Solid boxes indicate identical amino acids, and shaded boxes indicate conserved amino acids, for 50% of the sequences.

FIG. 2

Effect of a gerN mutation on OD loss during germination. (A) Germination of B. cereus 569 and gerN1 mutant spore suspensions in response to 5 mM inosine plus 10 mM NaCl (circles and squares, wild-type and mutant spores, respectively) or 10 mM l-alanine plus 10 mM NaCl (diamonds and triangles, wild-type and mutant spores, respectively). (B) Germination of B. cereus and gerN1 mutant spore suspensions in response to 50 μM inosine plus 10 mM NaCl (circles and squares, wild-type and mutant spores, respectively) or 50 μM l-alanine plus 10 mM NaCl (diamonds and triangles, wild-type and mutant spores, respectively).

FIG. 3

Germination of spore suspensions of B. cereus 569 (squares) and the gerN mutant (triangles) in calcium DPA.

FIG. 4

(A) Electron micrograph of B. cereus gerN1 mutant spores after germination in 5 mM inosine and 10 mM NaCl for 90 min. (B) Electron micrograph of B. cereus gerN1 mutant spores after germination and outgrowth in NB for 210 min. Bar, 1 μm.