- Volume 135, Issue 10, 1989

Germination at 37C of spores of Bacillus subtilis 1604 in the l-alanine and potassium phosphate (ALA) and the glucose, fructose, l-asparagine, potassium chloride (GFAK) germinant systems was triggered following heat activation at 70C for 1 h. In these conditions, 50% of the spore population became committed to germinate after exposure for 10 min and 14 min to ALA and GFAK, respectively, at which time 38% and 30% losses of OD600 had taken place. Dipicolinic acid (DPA) release, loss of heat resistance and release of soluble hexosamine-containing fragments occurred after commitment and were closely associated with loss of refractility in both the ALA and GFAK pathways. Net ATP synthesis could not be detected until 3–4 min after initiation of germination in both ALA and GFAK, by which time > 20% of the spore population was committed to germinate. The ALA and GFAK germination pathways were > 99% inhibited by 3 and 1 mm-HgCl2, respectively, as measured by OD600 loss. Reversible cost-commitment HgCl2-sensitive sites were present in the ALA and GFAK pathways which were 50% inhibited by 0·125 mm and 0·05 mm-HgCl2, respectively. A pre-commitment HgCl2-sensitive site was identified in the ALA pathway which was 55% inhibited by 6 mm-HgCl2. At 3 mm-HgCl2, 70% of the spore population became committed to germinate in the ALA pathway, whereas < 5% OD600 loss occurred. In this system, loss of heat resistance was associated with commitment, whereas OD600 loss and DPA release were identified as post-commitment events. The ALA and GFAK pathways were insensitive to a variety of metabolic inhibitors. Protease inhibitors had different effects on the ALA and GFAK pathways: phenylmethanesulphonyl fluoride (PMSF) solely inhibited ALA germination at a pre-commitment site and had little effect on GFAK germination, whereas N α-p-tosyl-l-arginine methyl ester (TAME) inhibited both the ALA and GFAK pathways at pre-and post-commitment sites. These results are discussed in relation to a recently proposed model for the triggering of Bacillus megaterium KM spore germination.

To cure the fireblight pathogen Erwinia amylovora of a 29 kb plasmid (pEA29) that is common in strains of these bacteria, restriction fragments of this plasmid were inserted into plasmids based on replication functions of bacteriophage fd, which cannot replicate in bacteria without expression of viral gene 2-protein. A 4·4 kb PstI fragment including the unique BamHI site of pEA29 allowed pfd plasmids to be propagated in E. amylovora. Furthermore, selection for these plasmids removed the natural 29 kb plasmid, apparently because of plasmid incompatibility. The pfd plasmids with the 4·4 kb insertion had a high tendency to segregate in E. amylovora when grown without selective pressure. Plasmid-free E. amylovora strains were virulent in standard tests on slices of immature pears, but symptom development was delayed compared to the wild-type. When assayed on pear seedlings a deficiency in pathogenicity was observed. Furthermore, strains without pEA29 spread more slowly on a lawn of pear cells than did wild-type strains. Plasmid-free strains of E. amylovora were not auxotrophic, but synthesis of extracellular polysaccharide was altered under certain growth conditions.

Twenty-five strains resembling ‘Streptococcus milleri’ were compared by DNA-DNA hybridization, by whole-cell-derived polypeptide patterns on SDS-PAGE, and by biochemical tests. Four homology groups were revealed by DNA-DNA hybridization. DNA homology groups 1, 2 and 3 were closely related and contained the type strains NCDO 2226 (Streptococcus constellatus), NCDO 2227 (Streptococcus intermedius) and NCTC 10713 (Streptococcus anginosus), respectively. DNA homology group 4 consisted of four strains received as variants of Streptococcus intermedius which were found not to be closely related to strains in groups 1-3. The data from SDS-PAGE polypeptide patterns and biochemical tests supported the recognition of three centres of variation within the ‘Streptococcus milleri group’ corresponding to DNA homology groups 1–3 and indicated that strains of DNA homology group 4 are members of an as yet undescribed species within the viridans streptococci.