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-ornothing phenomenon; these findings also suggest that damage to one or more key spore proteins causes spore killing by moist heat.Spores of Bacillus and Clostridium species formed in sporulation are metabolically dormant and extremely resistant to a variety of stress factors, including moist heat, dry heat, UV and gamma radiation, desiccation, and many toxic chemicals (18,32). Since spores of many of these species are commonly present in foodstuffs and since cells of some species can cause food spoilage or food-borne disease, much effort is expended in eliminating spores from foods. Moist heat is used routinely for inactivation of spores, generally at temperatures of Ն100°C for short to moderate periods of time. This method has been used for many, many years and is the gold standard for inactivation of spores in a food product.In general, spores are resistant to moist-heat temperatures that are ϳ45°C higher than those that inactivate growing cells of the same organism (36). A number of factors are responsible for spore moist-heat resistance, including the following: (i) the optimum growth temperature of the bacterial strain and the sporulation temperature (higher optimum growth and sporulation temperatures result in more resistant spores), (ii) the spore core's high level of dipicolinic acid (DPA) and its associated divalent cations, (iii) the type of divalent cations associated with DPA, (iv) the protection of spore DNA by its saturation with a group of ␣/␤-type small, acid-soluble spore proteins, and (v) the low water content in the spore core, which may contain as little as 25% of its wet weight as water in the most resistant spores (9,18,32).Even though the mechanisms of spore resistance to moist heat are fairly well understood, there is only a rudimentary understanding of the mechanism whereby spores are killed by this treatment, although this is not by DNA damage, since spore DNA is well protected by its saturation with ␣/␤-type small, acid-soluble spore proteins (18, 32). Moist-heat-treated spores often appear injured, and although they can be reco...

The study of individual patterns of longitudinal growth of the bony pelvic complex reveals the following findings concerning the sex differences in the growth of the bony pelvis.(1) The patterns of growth show the same individual variability and male-female overlap as the adult configuration of pelves.(2) The sex differences in the adult bony pelvis cannot be attributed to the differential response of one bone to sex hormone.(3) Sex differences develop from complicated variations in rates and direction of growth of local areas of the pelvic complex.( 4 ) The superior functional division of the bony pelvis shows only one notable sex difference -the sexual dimorphism of the directional growth of the anterior one-half of the iliac crest.(5) The inferior functional division of the bony pelvis shows numerous local areas of sexually dimorphic growth, but the major sex differences result from the greater lateral migration of the ischia.( 6 ) Of the regions showing definite sexual dimorphism in growth, the pelvic inlet and sciatic notch are the more variable because of their dependency on two separate anatomical systems for their final adult morphological configuration. The subpubic angle and length of the superior pubic ramus are directly associated with only one anatomical system, the enlargement of the pelvic cavity, thus, they show less variability and more definitive sex difference.

Aims:  To determine the mechanism of wet heat killing of spores of Bacillus cereus and Bacillus megaterium. Methods and Results:  Bacillus cereus and B. megaterium spores wet heat‐killed 82–99% gave two bands on equilibrium density gradient centrifugation. The lighter band was absent from spores that were not heat‐treated and increased in intensity upon increased heating times. These spores lacked dipicolinic acid (DPA) were not viable, germinated minimally and had much denatured protein. The spores in the denser band had viabilities as low as 2% of starting spores but retained normal DPA levels and most germinated, albeit slowly. However, these largely dead spores outgrew poorly if at all and synthesized little or no ATP following germination. Conclusions:  Wet heat treatment appears to kill spores of B. cereus and B. megaterium by denaturing one or more key proteins, as has been suggested for wet heat killing of Bacillus subtilis spores. Significance and Impact of the Study:  This work provides further information on the mechanisms of killing of spores of Bacillus species by wet heat, the most common method for spore inactivation.

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 ⌬vncS, ⌬vex3, ⌬vncR, and ⌬pep27 mutants, but not a ⌬lytA strain, to vancomycin were indistinguishable from that of the parent strain. ⌬vncS 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 ⌬vncS strain, in the parent strain R6, and in two other strains bearing erythromycin resistance markers, namely, a ⌬vncR strain and an unrelated ⌬comD 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 ␤-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.

Aims:  To determine conditions for generation and recovery of Bacillus subtilis spore populations heavily damaged by moist heat treatment. Methods and Results:  Bacillus subtilis spores were treated with moist heat and spore viability was assessed on different media. A rich medium and several minimal media gave similar spore recoveries after moist heat treatment, but lack of glucose in minimal media greatly decreased spore recovery. High NaCl levels also greatly decreased the recovery of moist heat‐treated spores on minimal media, and addition of good osmoprotectants reversed this effect. Moist heat treatment did not decrease spore recovery on minimal media with high salt through DNA damage or by eliminating spore germination, but by affecting spore outgrowth. Conclusions:  Conditions for generating B. subtilis spore populations with high levels of conditional moist heat damage have been determined. The major conditional damage appears to be in spore outgrowth, perhaps because of damage to one or more important metabolic enzymes. Significance and Impact of the Study:  This work has provided new insight into the mechanism of B. subtilis spore killing by moist heat.