sine plus high levels of DAP (3); growth with lower levels of DAP produced refractile spores with less cortex which were viable but heat sensitive (4). These data ...
JOURNAL OF BACTERIOLOGY, Feb. 1982, p. 494-498
Vol. 149, No. 2
0021-9193/82/020494-05$02.000/
Noninvolvement of the Spore Cortex in Acquisition of LowMolecular-Weight Basic Proteins and UV Light Resistance During Bacillus sphaericus Sporulation BARBARA SETLOW, REBECCA HAWES HACKETT, AND PETER SETLOW* Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032
Received 14 July 1981/Accepted 12 October 1981
Two major low-molecular weight, acid-soluble proteins (termed A and B proteins) were purified from Bacillus sphaericus spores and had properties similar to those of the analogous proteins from spores of other Bacillus species. These proteins were accumulated late in sporulation, when the developing spores became resistant to UV light, and were degraded during spore germination by a spore protease. A mutant ofB. sphaericus unable to make spore cortex because of a block in diaminopimelic acid (DAP) biosynthesis accumulated and maintained levels of the A and B proteins similar to those in the DAP+ parent or the DAPstrain in which cortex formation was restored by growth with DAP. In addition, the DAP- strain grown without DAP acquired a level of UV light resistance identical to that of wild-type spores and at the time of appearance of the A and B proteins. These findings indicate that formation of little, if any, spore cortex is required for acquisition of UV light resistance or maintainence of high levels of A and B proteins. The data provide further support for a role of the A and B proteins in the spore's UV light resistance.
Dormant spores of a number of Bacillus species contain high levels of two to three lowmolecular-weight, acid-soluble proteins (termed proteins A, B, and C in Bacillus megaterium) (10, 11, 16). These proteins are synthesized during sporulation as the developing spore becomes resistant to UV light (but 2 to 3 h before acquisition of heat resistance), and the majority are bound to spore DNA in vivo (1, 9, 10). The proteins are stable in the maturing forespore and in the dormant spore, but are degraded in the first minutes of spore germination (10, 14). This proteolysis generates many of the amino acids needed for protein synthesis early in spore germination, and the degradation is initiated by a spore endoprotease which is specific for the A, B, and C proteins (12, 13). The latter enzyme is also present in the developing forespore at the time of A, B, and C protein synthesis, but the protease does not attack its substrates (14). Two unanswered questions concerning this proteolytic system are: (i) do the A, B, and C proteins play a causal role in spore UV resistance; and (ii) how is the spore protease regulated such that it does not act in the developing forespore or dormant spore, yet acts rapidly in the first minutes of spore germination? Of particular interest to those studying properties of bacterial spores such as heat and UV resistance or dormancy is the role of unique structural components of the spore in such prop-
erties. For example, most explanations of spore heat resistance invoke a key role for the spore cortex-a peptidoglycan layer surrounding the core or central region of the spore (5, 15). Spore cortex peptidoglycan is similar in structure to vegetative cell wall peptidoglycan, but differs from it in some specific aspects including the presence in cortex (but not vegetative cell) peptidoglycan of muramic acid lactam (15). Similarly, spore cortex peptidoglycan of some species, including Bacillus sphaericus, contains diaminopimelic acid (DAP), whereas vegetative cell peptidoglycan contains lysine (3, 15). Imae and Strominger utilized this feature of B. sphaericus to study the involvement of spore cortex in specific spore properties by using mutants blocked in DAP biosynthesis (3). These mutants grew normally when supplemented with lysine, but produced no spore cortex (3, 4). The cortexless spores were not refractile in the phasecontrast microscope, accumulated little dipicolinic acid (DPA), and were not viable. Refractile, heat-resistant spores containing high cortex and DPA levels were produced by growth with lysine plus high levels of DAP (3); growth with lower levels of DAP produced refractile spores with less cortex which were viable but heat sensitive (4). These data suggested an essential role for spore cortex in the spore's acquisition of DPA, heat resistance, and refractility. Consistent with the latter data is work on the kinetics of 494
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ROLE OF SPORE CORTEX IN UV RESISTANCE
synthesis of spore cortex which indicates that cortex synthesis begins before acquisition of spore refractility, DPA, and heat resistance (2, 3, 5). However, these data are not sufficiently precise to allow determination of the kinetic relationship of cortex synthesis to earlier events in sporulation. Consequently, in this study we have utilized one of the DAP- mutants of B. sphaericus to investigate the possible role of spore cortex in spore UV resistance and the accumulation of the low-molecular-weight, acidsoluble proteins by the developing spore.
MATERIALS AND METHODS Bacterial strains and chemicals. The wild-type strain of B. sphaericus 9602 and its Lys- (strain 20-1 [3]) and DAP- (strain 32-3 [3]) derivatives were generously provided by Yasuo Imae (Institute of Molecular Biology, Nagoya University, Nagoya, Japan). Growth and isolation of spores and spore germination. B. sphaericus wild-type or mutant strains were grown at 33°C in SP medium containing thiamine (2.5 Ijg/ml), biotin (0.5 ,ug/ml), and lysine (100 ,ug/ml) with or without DL-DAP (1 mg/ml) as described previously (3). Cleaned dormant spores were prepared by isolation of sporulating cells 4 to 5 h after the spores had become refractile, use of lysozyme to complete sporangial lysis (3), and washing with water. These spore preparations contained
