INTRODUCTION

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Under certain nutrient stresses Bacillus species undergo a complex differentiation process resulting in the formation of highly resistant endospores, which subsequently, when favorable growth conditions return, germinate back to vegetative cells and grow and divide. The molecular genetics of sporulation (7, 25) and germination (14, 15) of Bacillus species have been reviewed.

Analysis of Bacillus subtilis mutants which are defective in response to one or both of their germinantsL-alanine or asparagine with glucose, fructose, and K⁺ (AGFK)suggests that the germinants interact with separate germinant-specific receptor complexes within the spore (14). Mutations within the gerA operon of B. subtilis specifically block germination initiated by L-alanine (34). The predicted amino acid sequences of the three GerA proteins encoded by the gerA operon suggest that they may be membrane associated, and they are the most likely candidates for the germinant receptor for L-alanine. Mutations in the gerB operon (18), responsible for AGFK germination in B. subtilis, allowed recognition of the novel germinant D-alanine; this strongly reinforces the argument that such gerA homologues encode the germinant receptor complexes.

B. cereus endospores have been shown to germinate in response to inosine and L-alanine; a combination of these germinants elicits the most rapid response (32). Operons encoding putative receptor complexes for the germinants have been named gerI (6) and gerL (3), respectively, and these operons are members of the gerA family identified in B. subtilis. How the germinant receptors are activated and how this leads to the global changes of spore germination have, however, not been determined.

Cation transport may play an important role in spore germination; a rise in the internal pH of germinating spores and release of Na⁺ and K⁺ have been shown to be early events, preceding dipicolinic acid (DPA) release. At least 80% of the spore's Na⁺ and K⁺ is released early in spore germination, the K⁺ being subsequently reabsorbed by an energy-dependent process (27).

Tani et al. (28) showed that a NaH antiporter homologue, named grmA, was important for the germination of spores of B. megaterium ATCC 12872 in any of its germinants (glucose, L-proline, L-leucine, or KNO₃). The mutant spores appeared to be blocked at an early stage of germination, as they did not lose heat resistance or release DPA, both relatively early events in spore germination. GrmA has a 47% amino acid identity to NapA of Enterococcus hirae (33), which has been shown to mediate NaH antiport activity (26). It was proposed that GrmA plays a critical role in the early stages of spore germination because of an effect on cation transport.

In this study a homologue of grmA was identified in B. cereus. The gene was designated gerN due to the effects of its disruption on spore germination. The gerN gene was shown to be important for the germination of B. cereus 569 UM20.1 spores in response to inosine, but not to L-alanine at optimal concentrations.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and media. Escherichia coli and B. cereus were routinely cultured in or on L broth and L agar containing the appropriate antibiotics (for E. coli, ampicillin at 50 µg ml¹; for B. cereus, erythromycin and lincomycin at 1 and 25 µg ml¹, respectively). CCY medium (24) was used for spore preparation. Nutrient agar no. 1 (NA; Oxoid) was used for spore enumeration. Some germination and outgrowth experiments were carried out in Oxoid nutrient broth (NB).

Spore preparation. A culture of B. cereus in CCY broth (700 ml inoculated with 7 ml of a mid-log-phase PAB [Penassay broth] culture) was incubated with shaking, at 37°C for 2 days, until>90% free spores were present. Spores were harvested and washed 10 times by repeated centrifugation and resuspension in distilled water, discarding the upper layer of cellular debris in the pellet from early washing steps. The spores were stored in distilled water at 20°C. Synchronous sporulation was carried out by the procedure of Sterlini and Mandelstam (22).

Spore germination assays. Spores were activated by heating in distilled water at 70°C for 30 min prior to germination.

OD fall. Spores were resuspended in germination buffer (10 mM Tris-HCl [pH 8.4]-10 mM salts) at an optical density at 580 nm (OD₅₈₀) 0.5. For L-alanine germination, 5 µg of O-carbamyl-D-serine ml¹ was added to inhibit L-alanine racemase activity. Germination was carried out at 37°C in inosine and at 30°C in L-alanine. After a 15-min preincubation of spores with buffer, germination was initiated by the addition of inosine (to 5 mM or 50 µM) or L-alanine (to 10 mM or 50 µM). The OD₅₈₀ of the spore suspension was monitored, and the percentage of the initial OD lost was calculated. Maximum rates of OD loss per minute quoted in the text were derived from the steepest part of the germination curve. Germination in calcium DPA was carried out by the method of Keynan and Halvorson (10).

DPA release. Heat-shocked spores were germinated at 1 mg (dry weight) ml¹. At intervals, 3-ml samples were removed and filtered (with a Millipore 0.45-µm-pore-size filter), and the filtrate was stored on ice. The DPA content of the filtrates was determined by a modification of the method of Janssen et al. (9).

Heat resistance loss. Spores were germinated in L-alanine or inosine. Samples (100 µl) were removed at intervals, serially diluted 100-fold to 10⁴ in sterile distilled water, preheated to 70 or 80°C, and incubated for up to 40 min at this temperature. Aliquots were plated in 3-ml overlays on NA plates. After overnight incubation at 37°C, colonies were counted and compared with a control prepared using spores that were not exposed to germinant but were subjected to heat treatment.

Measurement of ions released from germinating spores. Spores were heat activated and then washed 3 times with distilled water to remove any released ions. They were then germinated, at 10 mg (dry weight) of spores ml¹. No salts were added externally; samples were taken at intervals and pelleted by centrifugation (21,000 × g for 2 min at 4°C), and the ion content of the supernatant was assayed by atomic absorption spectrophotometry. Ions were released from dormant spores by autoclaving spores (1 mg [dry weight] per ml) in a total volume of 5 ml.

Measurement of ATP produced by germinating spores. Spores were germinated in 5 mM inosine. At intervals a 50-µl sample of the germinating spores was removed, added to 100 µl of lysis solution (Celsis International, Cambridge, United Kingdom), and incubated at room temperature for 3 min. Then 100 µl of luciferase-luciferin (10 mg ml¹; Sigma) was added to the sample, and the luminescence was measured for 10 s in a Celsis Opticomp luminometer.

Outgrowth of germinating spores. Germination and outgrowth were carried out in NB or, alternatively, the spores were germinated in Tris-HCl buffer (pH 8.4) plus L-alanine (10 mM) or inosine (5 mM), and then 2 volumes of NB were added to stimulate outgrowth. Germination and outgrowth were monitored by OD₅₈₀ measurement and phase-contrast microscopy.

Molecular biology methods. Electroporation of B. cereus was performed by the procedure of Bone and Ellar (5). PCR and inverse PCR, were carried out by standard procedures (17), using High Fidelity Taq Extend (Boehringer Mannheim). DNA sequencing was performed with the BigDye Terminator cycle sequencing Ready Reaction kit (Applied Biosystems) and an Applied Biosystems DNA sequencer. The DNA sequence was analyzed and assembled by using the Staden suite of programs (21).

Nucleotide sequence accession number. The 1,733-bp region including B. cereus gerN that was completely sequenced on both strands has been submitted to GenBank (accession number AF246294).

	RESULTS AND DISCUSSION

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Identification and sequencing of gerN, a homologue of the B. megaterium grmA gene. Degenerate PCR primers were designed to internal regions of GrmA conserved in NapA, using a codon usage table for B. cereus. The regions used (4) were from residue 65 (MFLAGLE) for the forward primer and from residue 266 (YAVFVPVFFV) for the reverse primer. Primers were designed with EcoRI and BamHI restriction sites at each end. PCR on B. cereus 569 UM20.1 chromosomal DNA yielded a single fragment of the expected size. This fragment was cloned into the integrational vector pMUTIN4 (31), and the inserts in two clones were sequenced (the plasmid was named pMNAP). They both contained an identical open reading frame (ORF), which would encode a product with 42% identity to the corresponding region of the GrmA protein.

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View larger version (150K): [in this window] [in a new window]   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.

Disruption of the gerN gene. The PCR fragment internal to the gerN gene (corresponding to codons 65 to 275) cloned into pMUTIN4 was introduced into B. cereus by electroporation, and the transformants were selected on erythromycin and lincomycin. Two transformants were characterized; AM1419 and AM1420. Intergration of the plasmid by homologous recombination into the chromosome within the gerN gene was confirmed by Southern blotting and the gene disruption mutant was named gerN1. This allele would encode a truncated product lacking the final 112 amino acids of the 387-residue protein.

Characterization of the gerN1 mutant. (i) Growth and sporulation. There is no difference between the growth rates of the wild-type and mutant strains in either NB or NB plus 0.5 M NaCl (data not shown). The gerN1 mutant showed normal sporulation in CCY medium and synchronous sporulation experiments (data not shown).

(ii) Measurement of spore germination in inosine by OD fall. Wild-type B. cereus 569 UM20.1 spores germinate rapidly in 5 mM inosine plus 10 mM NaCl in Tris-HCl buffer (maximum rate, 18% OD loss/min) (Fig. 2); OD fall, DPA release, loss of heat resistance, and phase darkening of the spores were 90% complete 10 min after the addition of germinant. In contrast, the rate of germination of the gerN mutant was severely inhibited under these conditions (maximum rate, 0.55% OD loss/min) (Fig. 2). The OD of the mutant spore suspensions did eventually fall as low as that of the wild type, but only after 3 h. The mutation blocked spore germination at an early stage; after 90 min, only 50 to 55% of the potential OD fall had occurred, 55 to 60% of the total DPA had been released, and 55 to 60% of the mutant spores still retained their heat resistance (data not shown). Surprisingly, at this time ca. 90% of the spores had become phase dark. Heat resistance loss and DPA release therefore occur at approximately the same rate as OD fall, but it appears that phase darkening of these spores can occur without the completion of the other events.

View larger version (16K): [in this window] [in a new window]   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).

(iii) Other changes during inosine germination. The gerN mutant and wild-type dormant spores were found to contain similar levels of Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe²⁺, and Mn²⁺ (29). Calcium and magnesium release from wild-type spores germinating in inosine was complete in 10 min, whereas the equivalent release in the gerN mutant was delayed (Table 1). Potassium ions are released and readsorbed (27), so the concentration in the supernatant rises (to less than the real maximum) and then decreases; the kinetics of these fluxes were also delayed in the mutant.

(iv) Germination in L-alanine. When germinated in 10 mM L-alanine and 10 mM NaCl, the gerN mutant spores show no significant defect (Fig. 2). Changing the cation adjunct had very little effect on the germination rate of either the wild-type or mutant spores (reference 6; also data not shown). However, in suboptimal concentrations of L-alanine (50 µM), the germination rate of the mutant, measured as OD fall, was reduced nearly threefold compared to that of the wild type (Fig. 2). This is different from the slow germination seen in 5 mM inosine, because, although the rate of response to germinant was reduced, the spores showed the normal pattern of germination behavior (29).

(v) Germination in calcium DPA. The rate of germination of the gerN mutant in the nonnutrient germinant, calcium DPA, was the same as that of the wild type (Fig. 3). Hence, germination which does not use the germinant receptors is unaffected and does not appear to require the function of the GerN protein.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Germination of spore suspensions of B. cereus 569 (squares) and the gerN mutant (triangles) in calcium DPA.

(vi) Cortex degradation. The peptidoglycan composition of spores was analyzed during germination. After germination for 90 min in 10mM inosine and 10 mM NaCl, only primordial cell wall remains in germinated wild-type spores, whereas the gerN null mutant had 60 to 70% of its cortex remaining (A. Atrih, personal communication). The peptidoglycan fragments released into the medium from the gerN mutant spores had the same structure as those from the wild-type spores, though they were released in lower quantities.

(vii) Electron microscopy of germinated and outgrowing spores. After 90 min of germination in 5 mM inosine (Fig. 4), cortex degradation and swelling of the spore core were apparent in only 50% of the gerN mutant spores, which appeared as an asynchronous mixture of spores at different stages of germination.

View larger version (89K): [in this window] [in a new window]   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.

The gerN spore germination defect. Disruption of the gerN gene of B. cereus 569 causes a major defect in inosine germination of the mutated spores. The normal germination process is blocked at an early stage, before loss of heat resistance; instead, there is a slow, residual germination response, which is asynchronous and grossly abnormal. As the gerN gene product is not required for germination in high concentrations of the alternative germinant L-alanine, an alternative protein must be able to carry out this or some analogous role. At limiting concentrations of L-alanine, the germination rate of mutant spores is slowed, suggesting GerN's involvement, at least under these conditions.