Fluorescence microscopic examination coupled with digital videoimage analysis of 4',6-diamidino-2-phenylindole-stained sporulating cells of Bacillus megaterium or Bacillus subtilis revealed a striking condensation of the forespore nucleoid. While both mother cell and forespore compartments had equal amounts of DNA, the forespore nucleoid became >2-fold more condensed than the mother cell nucleoid. The condensation of the forespore nucleoid began after only the first hour of sporulation, 2 to 3 h before expression of most forespore-specific genes including those for small, acid-soluble spore proteins, and was abolished in spoO mutants but not in spoIl or spoIll mutants. It is possible that this striking condensation of forespore DNA plays some role in modulating gene expression during sporulation.Sporulation in Bacillus species is characterized by the appearance of a number of new gene products at defined times during this differentiation process. This temporal pattern of gene expression is regulated in large part by ordered changes in the specificity of the cell's RNA polymerase through alterations in the pattern of associated sigma factors (10,12). In addition to temporal control of gene expression, sporulation is characterized by spatial control of gene expression. A key event early in sporulation is the appearance of an asymmetric septum dividing the cell into large and small compartments, with the small cell destined to become the forespore which will be engulfed by the larger mother cell. As would be expected, given the morphological difference between mother cell and forespore compartments, there are different patterns of gene expression in the two compartments (20,23). The differential gene expression in these compartments is also due, at least in part, to differences in RNA polymerase sigma factors, with one foresporespecific sigma factor (@rG) and one mother cell-specific sigma factor (0rK) identified to date (12,23,25). While this difference in sigma factors may explain much of the differential gene expression in the two compartments after the third hour (t3) of sporulation, it is not clear how this compartmentspecific difference in sigma factors is established, i.e., how compartment-specific gene expression is initiated. One formal possibility is that there is some difference in the structure of the genomes in the forespore and mother cell. Surprisingly, this possibility has not been considered in recent years, despite early evidence, primarily from light microscopy, that the forespore nucleoid becomes significantly condensed during sporulation (15,16,28,29). While forespore nucleoid condensation was clearly an early event in sporulation, this early work unfortunately did not precisely place the change within the time framework of the overall differentiation process, in particular with respect to synthesis of various sporulation-specific gene products. Consequently, we undertook an analysis of gross nucleoid structure during sporulation by using fluorescence microscopy of 4',6-diamidino-2-phenylindole...

Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination. In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by -0.4 during sporulation. By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6) (25). A second parameter which may be involved in spore dormancy is the pH in the spore core, which is rather low, 6.3 for Bacillus cereus spores and 6.4 for Bacillus megaterium spores, when internal pH is measured by distribution of the weak base methylamine (23). In another study, the internal pH of spores of Bacillus subtilis was found to be -6 when measured by 31P nuclear magnetic resonance spectroscopy (1). Although the internal pH of the dormant spore is rather low, it rises about 1 pH unit during spore germination (23,30 (20). The time of entry into sporulation is defined as to, with subsequent hours in sporulation noted as t1, t2, etc. At various times during sporulation, 1-ml samples were taken for assay of glucose dehydrogenase (GDH) and 2-to 4-ml samples were taken for measurement of DPA as previously described (19,20,27). In some cases the condensation of the forespore chromosome was assessed by staining glutaraldehyde-fixed sporulating cells with 4',6-diamidino-2-phenylindole and examining them through an epifluorescence microscope as described previously (22

Analysis of the pH decrease and 3-phosphoglyceric acid (3PGA) accumulation in the forespore compartment of sporulating cells of Bacillus subtilis showed that the pH decrease of 1 to 1.2 units at ϳ4 h of sporulation preceded 3PGA accumulation, as observed previously in B. megaterium. These data, as well as analysis of the forespore pH decrease in asporogenous mutants of B. subtilis, indicated that G -dependent forespore transcription, but not K -dependent mother cell transcription, is required for the forespore pH decrease. Further analysis of these asporogenous mutants showed an excellent correlation between the forespore pH decrease and the forespore's accumulation of 3PGA. These latter results are consistent with our previous suggestion that the decrease in forespore pH results in greatly decreased activity of phosphoglycerate mutase in the forespore, which in turn leads to 3PGA accumulation. In further support of this suggestion, we found that (i) elevating the pH of developing forespores of B. megaterium resulted in rapid utilization of the forespore's 3PGA depot and (ii) increasing forespore levels of PGM ϳ10-fold in B. subtilis resulted in a large decrease in the spore's depot of 3PGA. The B. subtilis strain with a high phosphoglycerate mutase level sporulated, and the spores germinated and went through outgrowth normally, indicating that forespore accumulation of a large 3PGA depot is not essential for these processes.

Small, acid-soluble proteins (SASP) of both the alpha/beta- and gamma-type were present in spores of Sporosarcina ureae and S. halophila, and three genes encoding alpha/beta-type SASP in these species have been cloned and sequenced. The amino acid sequences of the Sporosarcina alpha/beta-type SASP are extremely homologous to those of Bacillus SASP, further indicative of the close evolutionary relationship between these genera.

Laboratory technologists (22%) developed infections with Shigella sonnei. The isolates had the same antibiogram and pulse-field gel electrophoresis pattern as an unknown isolate handled by a laboratory student. Covering faucet handles with paper towels during hand washing in the laboratory was protective. No further cases occurred after the laboratory was cleaned with a phenolic agent and a handle-free faucet was installed.The incidence of infection acquired in hospital microbiology laboratories is approximately 4.0 per 1,000 person-years (9). Shigella species are among the many pathogens acquired in this setting (2)(3)(4)(5)9), and the risk of medical technologists acquiring shigellosis in clinical microbiology laboratories is approximately 0.84 per 1,000 person-years (4). This report describes an outbreak of Shigella sonnei infection among medical technologists in a hospital microbiology laboratory.(This work was presented in part at the 7th Annual Scientific