Analysis of the effects of a gerP mutation on the germination of spores of Bacillus subtilis.

As previously reported, gerP Bacillus subtilis spores were defective in nutrient germination triggered via various germinant receptors (GRs), and the defect was eliminated by severe spore coat defects. The gerP spores' GR-dependent germination had a longer lag time between addition of germinants and initiation of rapid release of spores' dipicolinic acid (DPA), but times for release of >90% of DPA from individual spores were identical for wild-type and gerP spores. The gerP spores were also defective in GR-independent germination by DPA with its associated Ca(2+) divalent cation (CaDPA) but germinated better than wild-type spores with the GR-independent germinant dodecylamine. The gerP spores exhibited no increased sensitivity to hypochlorite, suggesting that these spores have no significant coat defect. Overexpression of GRs in gerP spores did lead to faster germination via the overexpressed GR, but this was still slower than germination of comparable gerP(+) spores. Unlike wild-type spores, for which maximal nutrient germinant concentrations were between 500 µM and 2 mM for l-alanine and ≤10 mM for l-valine, rates of gerP spore germination increased up to between 200 mM and 1 M l-alanine and 100 mM l-valine, and at 1 M l-alanine, the rates of germination of wild-type and gerP spores with or without all alanine racemases were almost identical. A high pressure of 150 MPa that triggers spore germination by activating GRs also triggered germination of wild-type and gerP spores identically. All these results support the suggestion that GerP proteins facilitate access of nutrient germinants to their cognate GRs in spores' inner membrane.

Extremely variable conservation of γ-type small, acid-soluble proteins from spores of some species in the bacterial order Bacillales.

γ-Type small, acid-soluble spore proteins (SASP) are the most abundant proteins in spores of at least some members of the bacterial order Bacillales, yet they remain an enigma from both functional and phylogenetic perspectives. Current work has shown that the γ-type SASP or their coding genes (sspE genes) are present in most spore-forming members of Bacillales, including at least some members of the Paenibacillus genus, although they are apparently absent from Clostridiales species. We have applied a new method of searching for sspE genes, which now appear to also be absent from a clade of Bacillales species that includes Alicyclobacillus acidocaldarius and Bacillus tusciae. In addition, no γ-type SASP were found in A. acidocaldarius spores, although several of the DNA-binding α/ß-type SASP were present. These findings have elucidated the phylogenetic origin of the sspE gene, and this may help in determining the precise function of γ-type SASP.

How moist heat kills spores of Bacillus subtilis.

Populations of Bacillus subtilis spores in which 90 to 99.9% of the spores had been killed by moist heat gave only two fractions on equilibrium density gradient centrifugation: a fraction comprised of less dense spores that had lost their dipicolinic acid (DPA), undergone significant protein denaturation, and were all dead and a fraction with the same higher density as that of unheated spores. The latter fraction from heat-killed spore populations retained all of its DPA, but >/=98% of the spores could be dead. The dead spores that retained DPA germinated relatively normally with nutrient and nonnutrient germinants, but the outgrowth of these germinated spores was significantly compromised, perhaps because they had suffered damage to some proteins such that metabolic activity during outgrowth was greatly decreased. These results indicate that DPA release takes place well after spore killing by moist heat and that DPA release during moist-heat treatment is an all-or-nothing phenomenon; these findings also suggest that damage to one or more key spore proteins causes spore killing by moist heat.

Vancomycin tolerance induced by erythromycin but not by loss of vncRS, vex3, or pep27 function in Streptococcus pneumoniae.

Vancomycin-tolerant Streptococcus pneumoniae is a growing problem among drug-resistant human pathogens. Some vancomycin-tolerant pneumococci have been reported to carry mutations in loci encoding a two-component regulatory system designated VncRS or in a proximal ABC transporter, Vex. A model was advanced proposing that the tolerance phenotype resulted from the inability of a vncS mutant to respond to the Vex-transported Pep27 "death peptide" signal and dephosphorylate VncR, thereby preventing relief of repression of autolytic and other cell death functions in response to antibiotics. To explore this hypothesis, we constructed mutations in vncS, vncR, vex3, and pep27 in S. pneumoniae strain R6 and two additional genetic backgrounds. The lytic responses of the isogenic DeltavncS, Deltavex3, DeltavncR, and Deltapep27 mutants, but not a DeltalytA strain, to vancomycin were indistinguishable from that of the parent strain. DeltavncS strains also failed to exhibit tolerance to vancomycin at various doses in multiple media and showed wild-type sensitivity to other classes of autolysis-inducing antibiotics. In contrast, addition of subinhibitory levels of the antibiotic erythromycin led to tolerance to vancomycin during late, but not early, exponential-phase growth in a DeltavncS strain, in the parent strain R6, and in two other strains bearing erythromycin resistance markers, namely, a DeltavncR strain and an unrelated DeltacomD strain that is defective in competence-quorum sensing. Thus, this tolerance effect resulted from changes in cell growth or other erythromycin-dependent phenomena and not inactivation of vncS per se. Consistent with these results, and in contrast to a previous report, we found that a synthetic form of Pep27 did not elicit lytic or nonlytic killing of pneumococci. Finally, microarray transcriptional analysis and beta-galactosidase reporter assays revealed VncS-dependent regulation of the vex123 gene cluster but did not support a role for VncRS in the regulation of autolytic or other putative cell death loci. Based on these findings, we propose that vancomycin tolerance in S. pneumoniae does not result from loss of vncS function alone.