The outer coat fraction (OC-Fr) of Bacillus megaterium ATCC 12872 spore was isolated as a resistant residue after alkali extraction, sonic treatment, and pronase digestion of the spore coat preparation, and its backbone structure was determined by chemical analysis to be composed of galactosamine-6-phosphate (GalN-P) polymers with polypeptides and calcium. OC-Fr was not fully solubilized after ordinary acid hydrolysis. OC-Fr was insensitive to all hexosaminidases tested, and moreover, an isolated fragment, a pentamer of GalN-P, was also resistant to lysozyme and hexosaminidases even after N-acetylation, being sensitive to them to some extent after dephosphorylation. Molecular sieving experiments revealed that the outer coat limited the entry of compounds with a molecular weight of more than 2,000. Exchange of the metal on the spore surface also influenced the heat resistance. Spores of OC-Fr-deficient mutants were less resistant but were still much more resistant than the vegetative cells. These results suggest that the outer coat protects the contents of the spore against chemical, physical and enzymatic treatments owing to the chemical structure itself, composed mainly of GalN-P polymers, and the molecular sieving effect.

Outermost layer deficient mutant spores of Bacillus megaterium ATCC 12872 were isolated by Urografin density gradient centrifugation after mutagenesis with ethyl methanesulfonate. Although the composition of the cortex peptidoglycan was the same as that of the parent spores, three major proteins (48, 36, and 22 K daltons) were missing, suggesting that these proteins are components of the outermost layer. All mutant spores were also found to have very hydrophobic surface by 'salt aggregation test,' which would facilitate selection of such mutants.The bacterial spores have chemically and morphologically specific structural components: the cortex, the coat, and the exosporium. Bacillus megaterium ATCC 12872 (QM B1551) spore has a thick outermost layer (OL) outside of the inner coat (IC) (2). This layer had been called "outer coat" by many including our group, but Gerhardt's group proposed to call it "exosporium" from its morphological and chemical features ( 11 ) . But it is not a typical exosporium as observed in B. cereus spores. We use the term "outermost layer" tentatively because terminology is not an essential problem in this paper. This layer is formed at the later stages of sporulation and is resistant to several treatments: sonic disintegration, lysozyme digestion, and alkali extraction (8). Our previous papers (15,16) showed that OL consists of a 'skelton' of phosphorylated galactosamine polymer carrying mannose in some parts and also contains peptides/proteins. But its function, biosynthesis, and especially morphogenesis, have not been clearly known. OL deficient mutant spore is a useful tool for elucidating these subjects. Koshikawa et al have isolated one OL deficient mutant (ATCC 33729) of this strain by chance selection (11), but only one mutant is not enough for this purpose so that we tried to isolate these mutant spores by taking advantage of the difference in density between the mutant and the parent spores.The parent strain, B. megaterium ATCC 12872 (QM B1551), was obtained from the American Type Culture Collection and the mutant, ATCC 33729, was supplied by Dr. Koshikawa at Setsunan University. According to the method of Ito and Spizizen (7) , the parent spores were treated with 0.45 M ethyl methanesulfonate 973

Decoated spores of Bacillus megaterium ATCC 12872 were prepared by extracting the inner coat components with an alkaline solution containing sodium dodecyl sulfate and dithiothreitol (SDS-DTT) from outer coat-deficient mutant spores, which were produced from one of the mutants isolated and named MAE-05 by us. The decoated mutant spores germinated as well as the intact spores of the mutant and the parent, indicating that the outer and inner spore cats cannot be essential structures for the initiation of germination.When the SDS-DTT-treated MAE-05 spores were converted to H-spores by incubation in citrate-phosphate buffer (pH 3.5) at 30 C for 3 hr, they lost their germinability by glucose and KNO3. Ca-spores, prepared by treating H-spores with 10 mm calcium acetate at 37 C for 60 min, regained the germinability . Experiments on the interaction of 45Ca with the cortex and the inner membrane isolated from H-spores suggested that the calcium present in the inner membrane might be related to germinability.

It was proved that three spore coat proteins of 48, 36, and 22 kDa (P48, P36, and P22) were the components of the outermost layer (OL) of Bacillus megaterium ATCC 12872 spore by analysis of the isolated OL. And it was indicated that these proteins were deposited not by disulfide bond, but by ionic and/or hydrophobic bonds on the spore. Among them, P36 and P22 were expected to be located on the very surface of the spore by immunological analysis. In the OL deficient mutant of B. megaterium ATCC 12872, MAE05, whose spore was lacking in these OL proteins and galactosamine-6-phosphate polymer, both P36 and P22 were present in the mother cell cytoplasm and deposited on the forespores, but they disappeared with the lysis of mother cells. An OL protein-releasing factor having proteolytic activity was detected in the culture supernatant at the late sporulating stage of both the wild-type and the mutant strains. But the factor could not act on the proteins of the mature spores and the forespores at tin (tn indicates n hr after the end of exponential growth) of the wild-type strain. Moreover, P36 and P22 were found in the spores of a revertant of MAE05 which could form galactosamine-6-phosphate polymer, suggesting that this sugar polymer played the role in protecting the OL proteins against the protease-like substance after the deposition.

We isolated two galactosamine derivatives from Bacillus megaterium sporulating cells by lectin affinity chromatography followed by DEAE-Sephadex A-25 chromatography. From chemical analyses and measurements of these compounds, it was determined that one was uridine 5'-diphospho-N-acetylgalactosamine and that the other was uridine 5'-diphosphogalactosamine. They appeared in the middle stage of sporulation and disappeared during the period when galactosamine-6-phosphate is deposited on the forespore surface. These results suggest that uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosaiine are intermediates in the biosynthesis of the galactosamine-6-phosphate polymer, a backbone structure of the exosporium.The exosporium, the outermost integument of the spore, is a spore-specific constituent and is found as an electrondense layer under an electron microscope. This structure is visualized at the later stages of spore formation as part of the sporulation process, and its morphogenesis must be under the control of gene expression during sporulation. To understand the regulatory mechanism of this morphogenetic event, it is necessary to know the biochemical changes associated with exosporium formation.It has been reported that galactosamine-6-phosphate (GalN-6-P) is a major component of the exosporium (previously called the outer coat by us) in Bacillus megaterium ATCC 12872 (4, 5). However, the biosynthetic pathway of the GalN-6-P polymer has not been determined. In another paper, we reported that the activity of uridine 5'-diphospho-N-acetylglucosamine-4-epimerase (UDP-GlcNAc-4-epimerase), which catalyzes the interconversion between uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) and uridine 5'-diphospho-N-acetylgalactosamine (UDPGalNAc), was found to increase at the middle stage of sporulation (4a). UDP-sugars are usually intermediates in the synthesis of sugar polymers, and galactosamine has been detected only in the exosporium (4). Accordingly, it is conceivable that galactosamine derivatives which combine with UDP can be the intermediates in the biosynthetic pathway of the GalN-6-P polymer.To obtain galactosamine derivatives, we used the lectin isolated from Bandeirea simplicifolia, which has a high affinity for a-linked D-galactopyranosyl and 2-acetoamido-2-deoxy-D-galactopyranosyl residues (3). Lectin I from B. simplicifolia seeds (Calbiochem-Behring) was prepared by a previously described method (2) and coupled to Sepharose 4B as described by Blake and Goldstein (1). Lectin affinity chromatography of the trichloroacetic acid-soluble fraction of sporulating cell (T7) cytoplasm was carried out by the method of Piller et al. (6) (Fig. la). Figure 2 shows the * Corresponding author.high-pressure liquid chromatography profile observed at 262 nm on the ion-pair mode of the above-described lectinadsorbed fraction. There were two major peaks, designated as unknown A and unknown B.These two compounds were purified by DEAE-Sephadex A-25 chromatography. After the lectin-adsorbed fraction was...