Abstract
Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3-C-methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM:C-methyltransferase, and NADPH-dependent CDP-3-C-methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3-C-methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3-C-methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C-methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3-C-methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus.
Highlights
Bacterial cells produce myriad glycan structures that are secreted or attached to cell surfaces
This is proposed to facilitate transport of the spores to sites of spore germination and bacterial outgrowth [10, 11]. These two glycoproteins characterized in the B. cereus ATCC 14579 exosporium have different sugar composition and glycosylation patterns [12]
Spores are decorated with two rare sugars, 3-C-methyl-6-deoxyhexoses
Summary
Bacterial glycan structures on cell surfaces are critical for cellcell recognition and adhesion and in host-pathogen interactions. Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. The BclA protein plays a central role in Bacillus anthracis pathogenesis by promoting the interaction of spores with the host phagocytic cell This is proposed to facilitate transport of the spores to sites of spore germination and bacterial outgrowth [10, 11]. Genome sequence analyses have revealed the presence of at least 13 different collagen-like protein-encoding genes, and proteomic studies indicate that some of them are in the exosporium [13] Whether these are glycoproteins with glycans that differ in their sugar composition and sequence remains unknown. The formation of the C-methyl-sugar-containing glycans on spore surface occurs when cells are induced to grow in a sporulation medium
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