Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants

Abstract

Bacillus cereus 569 (ATCC 10876) germinates in response to inosine or to L-alanine, but the most rapid germination response is elicited by a combination of these germinants. Mutants defective in their germination response to either inosine or to L-alanine were isolated after Tn917-LTV1 mutagenesis and enrichment procedures; one class of mutant could not germinate in response to inosine as a sole germinant but still germinated in response to L-alanine, although at a reduced rate; another mutant germinated normally in response to inosine but was slowed in its germination response to L-alanine. These mutants demonstrated that at least two signal response pathways are involved in the triggering of germination. Stimulation of germination in L-alanine by limiting concentrations of inosine and stimulation of germination in inosine by low concentrations of L-alanine were still detectable in these mutants, suggesting that such stimulation is not dependent on complete functionality of both these germination loci. Two transposon insertions that affected inosine germination were found to be located 2.2 kb apart on the chromosome. This region was cloned and sequenced, revealing an operon of three open reading frames homologous to those in the gerA and related operons of Bacillus subtilis. The individual genes of this gerI operon have been named gerIA, gerIB, and gerIC. The GerIA protein is predicted to possess an unusually long, charged, N-terminal domain containing nine tandem copies of a 13-amino-acid glutamine- and serine-rich sequence.

FIG. 1

Concentration dependence of spore germination in response to l-alanine (solid circles) or inosine (open circles) in standard buffer conditions at 37°C. Values given are means (error bars represent standard deviations) of three independent experiments on the same spore preparation.

FIG. 2

Effect of l-alanine on riboside germination response. Germination was initiated by the addition of 1 mM riboside (either inosine, guanosine, or adenosine), with (shaded bars) or without (solid bars) 1 mM l-alanine as indicated. Values given are means (error bars represent standard deviations) of three independent experiments.

FIG. 3

Effect of temperature (A) and pH (B) on the maximum rate of germination in inosine and l-alanine. Germination was initiated by the addition of either 5 mM inosine (solid circles) or 100 mM l-alanine (open circles).

FIG. 4

Map and cloning strategy for characterization of the gerI operon. The arrows indicate the positions of the genes identified within the sequenced region. Restriction sites for EcoRI (E), ClaI (C), and HindIII (H) and sites of transposon insertion are indicated. The precise sites of insertion are bases 1412 and 3634 in the GenBank sequence. The extent of cloned inserts in plasmids pINO1 and pINO5, recovered from the chromosome of the transposon insertion mutants, and that in plasmid pMOC6, isolated from a B. cereus genomic library, are indicated. Dotted lines mark the unsequenced regions, which are not shown to scale.

FIG. 5

Features of the gerI sequence. (A) Predicted start codon and ribosome binding sites (RBS) for the met and gerIA genes and a putative ς^G-dependent promoter; (B) comparison of the ς^G promoter consensus sequence to the potential gerI promoter sequence; (C) coding region and potential RBS at the start of gerIB; (D) coding region and potential RBS at the start of gerIC; (E) predicted stop codon of gerIC and a potential rho-independent transcription terminator.

FIG. 6

Repeated region at the start of the gerIA gene. The nine repeats of 39 bp and 13 amino acids are shown. The number at the start of each nucleotide sequence refers to the position on the GenBank sequence.