Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis

Abstract

Small, acid-soluble spore proteins SASP-oa, SASP-,, and SASP--y as well as a SASP-j-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The aJ$-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-y was somewhat excluded from the nucleoid. These different locations of oda,-type and -y-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light.Approximately 10% of the protein of dormant spores of Bacillus ,subtilis is composed of a group of small, acidsoluble spore proteins (SASP) (18). The SASP are synthesized in parallel during sporulation within the developing forespore and are rapidly degraded during spore germination, thus providing amino acids for protein synthesis during this period of development. Three proteins, termed SASP-a, SASP-1, and SASP-y, make up approximately 75% of the SASP pool. SASP-a and SASP-3 are almost identical in primary sequence, and there are a number of minor SASP with primary sequences similar to those of SASP-cx and SASP-P. In contrast, SASP-,y has a more different primary sequence, and there is only a single y-type SASP (18).Other than its role in providing amino acids for protein synthesis during spore germination, SASP--y appears to have no function in spores (6). However, the a/,B-type SASP also play a key role in the resistance of spores to UV light, as they are intimately involved in the modification of the UV photochemistry of spore DNA which is essential for spore UV resistance (11,12,14). While the. mechanism whereby a/p-type SASP affect spore DNA photochemistry is not known, it seems likely that it involves direct SASP-DNA interactions, and it is known that SASP are localiied within the spore core, the site of spore DNA (18). SASP can also bind to DNA in vitro (18). However, this binding is weak, and attempts to isolate spore DNA or spore nucleoids with significant associated SASP have failed (17). One study using intact Bacillus megaterium spores and UV-induced protein-DNA cross-linking did provide evidence that significant ao/-type SASP was associated with spore DNA in vivo (15). However, from this study it was not possible to determine what percentage of these SASP were DNA associated.Because of the limitations of these previous techniques, we decided to attempt to localize various SASP within B. subtilis spores and forespores by using immunoelectron microscopy. However, initial attempts to localize SASP in dormant spores by using this technique were unsuccessful, since the cross-linking agents used in fixation (paraformaldehyde with or without glutaraldehyde) did not penetrate dormant spores sufficiently to prevent SASP movement during subsequent steps. The lack of penetration of dormant spores by cross-linking agents is not surprising in light of * Corresponding author. what is known of dormant-spore permeability (5) as well as the resi...

Compartmentalization of Gene Expression duringBacillus subtilisSpore Formation

“…Direct evidence that the expression of particular genes was completely compartmentalized was obtained by the use of immunoelectron microscopy with antibodies to small acid-soluble proteins (SASPs), which revealed that these proteins are found exclusively within the prespore (83). The utility of this technique was expanded by using antibodies to ␤-galactosidase on samples from strains expressing spo-lacZ transcriptional fusions (48,189).…”

“…It was shown 25 years ago through a study of genetic mosaics that gene expression is compartmentalized during sporulation of B. subtilis, with different genes being expressed in the prespore and the mother cell, the two cell types involved (43,226). In the years since, the completeness of compartmentalization has been demonstrated first by immunoelectron microscopy (48,83,189), then by fluorescence microscopy with immunofluorescence and green fluorescent protein (105,170,233,299,314), and most recently through the use of a two-part transcriptional probe (173).…”

Characterization of bofA, a gene involved in intercompartmental regulation of pro-sigma K processing during sporulation in Bacillus subtilis

“…At intermediate to late stages of development, that is, after engulfment, gene expression in the forespore is directed by a transcription factor called or' (15,30), whereas gene expression in the mother cell is under the control of a transcription factor called o.K (16). The or' and or factors are bona fide cell-typespecific regulatory proteins in that both their synthesis and their sites of action are confined to one or the other compartment (9,12,15,17).…”

Immunogold localization of glutathione transferase B1‐1 in Proteus mirabilis

“…Cells of Proteus mirabihs were tixed according to Francesconi et al [20] with modifications as follows. Cells were centrifuged for 5 min and were fixed in (v/v) 2% paraformaldehyde/O.l% glutaraldehyde in 100 mM cacodylate buffer, pH 7.2, for 4 h at 4°C.…”