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.

An airlift fermentor system allowing precise regulation of pH and aeration combined with a chemically defined medium was used to study growth and sporulation of Bacillus cereus ATCC 14579. Sporulation was complete and synchronous. Expression of sigA, sigB, sigF, and sigG was monitored with real-time reverse transcription-PCR, and the pattern qualitatively resembled that of Bacillus subtilis. This method allows reproducible production of stable spores, while the synchronous growth and defined conditions are excellently suitable for further gene expression studies of cellular differentiation of B. cereus.

Bacillus cereus endospores germinate in response to particular nutrients. Spores are able to sense these nutrients in the environment by receptors encoded by the gerA family of operons. Analysis of the Bacillus cereus ATCC 14579 genome revealed seven gerA family homologues. Using a transposon Tn917-based insertional mutagenesis approach followed by an enrichment procedure to select for L-alanine-induced germination mutants, we isolated a mutant with a defect in the L-alanine germination pathway. The transposon disrupted the last gene of a tricistronic gerA family operon, designated gerR, with the order gerRA, gerRC, gerRB. A second mutant was created by insertion of pMUTIN4 in gerRC. Both mutants showed the same phenotype for nutrient-induced germination. Spores of the gerR mutant strains were blocked in their L-alanine-initiated germination pathway and showed a delayed inosine-induced germination response. Apparently, germination mediated by L-alanine and inosine cannot be compensated for completely by the other germinant receptors, and this points towards an essential role of the gerR-encoded receptor in the receptor complex. In food products, spores of the mutant strains showed a reduced germination response compared to spores of the parental strain. High-pressure-initiated germination was not affected by the gerR mutations, as experiments with 100 and 550 MPa showed no difference with spores of the parental strain.

Bacillus cereus ATCC 14579 endospores were produced in Y1 medium, a nutrient-rich, chemically defined sporulation medium, and in modified G medium, containing low amounts of nutrients. The average transcription level of the seven ger operons per cell was 3.5 times higher in Y1 medium, and the spores grown in this medium showed an enhanced germination response.Bacillus cereus is a gram-positive, rod-shaped food pathogen that can sporulate under nutrient-limiting conditions. The conditions under which sporulation takes place determine spore characteristics such as wet-heat resistance and germination properties (7,18,22,26). Once formed, spores can return to vegetative growth by an irreversible process called germination (19,21,24). Germination receptors, which are located in the inner membrane of the spore, play a crucial role in the initiation of germination, as spores lacking these receptors are strongly impaired in their responses to germinant molecules (20). The signaling and activation pathway(s) mediated by these receptors is still unknown. It has been shown, however, that spores contain different types of receptors that can respond to specific germinant molecules, mostly amino acids or purine ribonucleosides (1,4,10,11,13,19,27). Germination receptors are encoded by tricistronic operons called ger operons, and the three gene products are necessary to form a functional receptor (19). During sporulation, the ger operons are transcribed in the forespore by a sigma G-dependent RNA polymerase (21). The genome of B. cereus ATCC 14579 contains seven putative ger operons, which may equip the spore with a set of seven functional receptors (11). The Bacillus subtilis genome contains five ger operons, of which three have been characterized (5,14,28). These three ger operons were expressed at very low levels (5,8,28). The composition of the medium can affect various spore properties (3, 7, 9), but the effect of medium composition on the transcription of the ger operons and its impact on germination properties of the spores is not known. Changes in ger operon expression may cause variation in the number of receptors in the spore, which consequently affects the nutrient-induced germination properties.This report describes the transcriptional analysis of each of the seven ger operons of B. cereus ATCC 14579 during sporulation in nutrient-rich Y1 medium, containing approximately 30 mM amino acids and 10 mM glucose, and in modified G medium, containing approximately 14 mM amino acids and no glucose. The composition of the medium had a significant impact on expression of the B. cereus ger operons and the spores' nutrient-induced germination characteristics.Bacterial strains, spore preparation, and transcriptional analysis. The B. cereus ATCC 14579 ger mutant strains used were disrupted in each of the seven ger operons by the insertion of plasmid pMUTIN4 as described previously (11). For all insertions, the lacZ reporter gene present on pMUTIN4 was under the control of the ger operon promoter, facilitating the measurement of ...

A chemically defined medium in combination with an airlift fermentor system was used to study the growth and sporulation of Bacillus cereus ATCC 14579. The medium contained six amino acids and lactate as the main carbon sources. The amino acids were depleted during exponential growth, while lactate was metabolized mainly during stationary phase. Two concentrations of glutamate were used: high (20 mM; YLHG) and low (2.5 mM; YLLG). Under both conditions, sporulation was complete and synchronous. Sporulation started and was completed while significant amounts of carbon and nitrogen sources were still present in the medium, indicating that starvation was not the trigger for sporulation. Analysis of amino acids and NH 4 ؉ in the culture supernatant showed that most of the nitrogen assimilated by the bacteria was taken up during sporulation. The consumption of glutamate depended on the initial concentration; in YLLG, all of the glutamate was used early during exponential growth, while in YLHG, almost all of the glutamate was used during sporulation. In YLLG, but not in YLHG, NH 4 ؉ was taken up by the cells during sporulation. The total amount of nitrogen used by the bacteria in YLLG was less than that used by the bacteria in YLHG, although a significant amount of NH 4 ؉ was present in the medium throughout sporulation. Despite these differences, growth and temporal expression of key sigma factors involved in sporulation were parallel, indicating that the genetic time frames of sporulation were similar under both conditions. Nevertheless, in YLHG, dipicolinic acid production started later and the spores were released from the mother cells much later than in YLLG. Notably, spores had a higher heat resistance when obtained after growth in YLHG than when obtained after growth in YLLG, and the spores germinated more rapidly and completely in response to inosine, L-alanine, and a combination of these two germinants.Bacillus cereus is a gram-positive, facultative anaerobic rodshaped bacterium able to form spores. It is a ubiquitous bacterium found in soil and in many raw and processed foods, such as rice, milk and dairy products, spices, and vegetables (8,12,20,44). Many strains of B. cereus are able to produce toxins and cause distinct types of food poisoning (19,31). Concerns over B. cereus contamination have increased over the past few years because of the rapidly expanding market of chilled foods that may be pasteurized but still contain viable spores (8,20,34). Spores from B. cereus can germinate and outgrow during storage, even at low temperatures (8,11,20). To address this increasing problem, major efforts focus on determining the causes of spore resistance and the mechanisms of germination.It has been well established that bacterial spore properties are affected by the conditions during sporulation (1,17,18,33,41). In most studies, spores are routinely produced from fortified agar or rich liquid media, which results in heterogeneous sporulation conditions for the individual cells. This prevents careful analysis of t...