Occurrence in Bacillus megaterium of uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine, intermediates in the biosynthesis of galactosamine-6-phosphate polymer

Abstract

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...

Orthologues of Bacillus subtilis Spore Crust Proteins Have a Structural Role in the Bacillus megaterium QM B1551 Spore Exosporium

“…Finally, a number of earlier studies speculate that the exosporium of B. megaterium QM B1551 spores contains a galactosamine-6-phosphate polymer skeleton that confers rigidity to the structure (14,25,32). A number of genes that may have a role in the synthesis of such a polymer, including BMQ_pBM60014 and BMQ_pBM60015, are carried, typically in clusters, on plasmid pBM600.…”

Characterization and Deposition of the Proteins in the Outermost Layer of Bacillus megaterium Spore

“…We reported that galactosamine-6-phosphate polymer begins to be deposited on the forespore from t9 (17) and may be responsible for the surface hydrophobicity of the spore (22). In this study, it was indicated that this sugar polymer does not participate in the deposition of P36 and P22, but may play the role in protecting these OL proteins against the releasing factor after deposition, because loss of these proteins was not observed in the mature spore and the forespore at tio of the wildtype strain where the sugar polymer had been deposited.…”