Characterization of the Enzymes Encoded by the Anthrose Biosynthetic Operon of Bacillus anthracis

Abstract

Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-␤-L-rhamnose ␣-1,3-L-rhamnosyl transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD-encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit ␤-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N-acyltransferase activity.

Genetic and Biochemical Characterizations of Enzymes Involved in Streptococcus pneumoniae Serotype 2 Capsule Synthesis Demonstrate that Cps2T (WchF) Catalyzes the Committed Step by Addition of β1-4 Rhamnose, the Second Sugar Residue in the Repeat Unit

“…The Cps2L product was confirmed to be dTDP-Glc by its retention time, which was similar to that of the standard dTDP-Glc. The Cps2N and Cps2M reaction products had a delayed retention time compared to that of their precursors, similar to other dTDP-keto intermediates analyzed with these methods (16). The shorter retention time for the Cps2O product relative to dTDP-Glc is consistent with that reported for dTDPRha in other publications (23,50).…”

Antibody Responses to a Spore Carbohydrate Antigen as a Marker of Nonfatal Inhalation Anthrax in Rhesus Macaques

“…These data provide proof of concept for the antigenicity of the exosporium anthrose trisaccharide as a marker of B. anthracis spore exposure and infection. Future studies will evaluate enhanced analytic sensitivity of the anti-ATS antibody detection assay and determine the diagnostic sensitivity and specificity of the anti-ATS antibodies and their utility as a diagnostic tool (7,19). Collectively these data will contribute to our ability to quickly detect exposure to B. anthracis spores.…”

Identification of an African Bacillus anthracis Lineage That Lacks Expression of the Spore Surface-Associated Anthrose-Containing Oligosaccharide

“…In contrast, inactivation of gene BAS3322 resulted in spores with about half as much anthrose as found in wild-type spores (6). Furthermore it was reported that inactivation of gene BAS3321 resulted in BclA being substituted with trisaccharides only, suggesting that the enzyme is a dTDP-␤-L-rhamnose ␣-1,3-L-rhamnosy transferase that attaches the fourth residue of the pentasaccharide side chain (7). Inactivation of genes BAS3320 and BAS3319 resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose (7).…”