PubMed · 14832427
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T W PEARCE, E O POWELL. 1951. A selective medium for Bacillus anthracis.. https://doi.org/10.1099/00221287-5-2-387
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The molecular basis of endospore formation in the model gram-positive bacterium Bacillus subtilis has been investigated for over half a century. Here, using high throughput and classical genetic approaches, we performed a comparative analysis of sporulation in the human pathogen Bacillus anthracis. A transposon-sequencing screen identified >150 genes required for B. anthracis sporulation. As anticipated, many of the genes that are critical for sporulation in B. subtilis were also required for B. anthracis sporulation. However, we identified >50 genes that are important for sporulation in B. anthracis but not in B. subtilis, and 22 B. anthracis sporulation genes that are absent from the B. subtilis genome. To validate the hits from our screen, we generated an ordered transposon-mutant library using Knockout Sudoku. Cytological analysis of a subset of the canonical sporulation-defective mutants revealed similar but not identical phenotypes in the pathogen compared to the model. We investigated several of the newly identified sporulation genes, with an in-depth analysis of one, ORF 04167, renamed ipdA. Sporulating cells lacking ipdA are blocked in the morphological process of engulfment, generating septal bulges. An AlphaFold-Multimer screen and a classical genetic enrichment revealed that IpdA is a secreted inhibitor of the polysaccharide deacetylase PdaN. Our data support a model in which induction of IpdA at the onset of sporulation inhibits deacetylation of the cell wall peptidoglycan (PG), enabling the sporulation-specific PG hydrolases to catalyze engulfment. Altogether, our studies reveal that B. subtilis is an excellent model for endospore formation in B. anthracis, while underscoring the importance of direct analysis in B. anthracis. The suite of tools that we have generated will catalyze the molecular dissection of sporulation and other cell biological processes in this important human pathogen.
The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide-oligonucleotide-phycoerythrin conjugates ('burrs') that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins.
The beta glycoside of the tetrasaccharide sequence beta-Ant-(1-->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1-->2)-l-Rhap, whose aglycon allows conjugation to proteins, was synthesized for the first time. A stepwise synthetic approach was applied with thioglycosides as glycosyl donors, and the beta anomer of the compound was obtained equipped with a spacer group whose further transformation allows conjugation to suitable carriers. To synthesize the beta-anthrosyl linkage with high stereoselectivity, a linker-equipped rhamnotriose derivative was glycosylated with ethyl 4-azido-3-O-benzyl-2-O-bromoacetyl-4,6-dideoxy-1-thio-beta-d-glucopyranoside. Further functionalization of the tetrasaccharide thus obtained, followed by deprotection, gave the target substance.