Biosynthesis of a New UDP-sugar, UDP-2-acetamido-2-deoxyxylose, in the Human Pathogen Bacillus cereus Subspecies cytotoxis NVH 391-98

Abstract

We have identified an operon and characterized the functions of two genes from the severe food-poisoning bacterium, Bacillus cereus subsp. cytotoxis NVH 391-98, that are involved in the synthesis of a unique UDP-sugar, UDP-2-acetamido-2-deoxyxylose (UDP-N-acetyl-xylosamine, UDP-XylNAc). UGlcNAcDH encodes a UDP-N-acetyl-glucosamine 6-dehydrogenase, converting UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetyl-glucosaminuronic acid (UDP-GlcNAcA). The second gene in the operon, UXNAcS, encodes a distinct decarboxylase not previously described in the literature, which catalyzes the formation of UDPXylNAc from UDP-GlcNAcA in the presence of exogenous NAD ؉ . UXNAcS is specific and cannot utilize UDP-glucuronic acid and UDP-galacturonic acid as substrates. UXNAcS is active as a dimer with catalytic efficiency of 7 mM ؊1 s ؊1. The activity of UXNAcS is completely abolished by NADH but unaffected by UDP-xylose. A real-time NMR-based assay showed unambiguously the dual enzymatic conversions of UDP-GlcNAc to UDP-GlcNAcA and subsequently to UDP-XylNAc. From the analyses of all publicly available sequenced genomes, it appears that UXNAcS is restricted to pathogenic Bacillus species, including Bacillus anthracis and Bacillus thuringiensis. The identification of UXNAcS provides insight into the formation of UDP-XylNAc. Understanding the metabolic pathways involved in the utilization of this amino-sugar may allow the development of drugs to combat and eradicate the disease.A food poisoning outbreak in France in 1998 led to the isolation of a new strain of Bacillus cereus subsp. cytotoxis NVH 391-98 (1). This rod-shaped Gram-positive bacterium causes a disease that initially produces emetic (nausea and vomiting)-like symptoms, and/or in more severe cases produces a diarrheal form that causes abdominal cramps and diarrhea (2). Similar to its close relatives, the notorious human pathogen Bacillus anthracis and the insecticidal Bacillus thuringiensis, the B. cereus strain can form spores. Due to its cell surface, a spore can survive harsh conditions (e.g. soil and air), and when the environment becomes appropriate, it will germinate, resulting in a vegetative cell that can produce emetic toxin and different enterotoxins (3). The cell surfaces of many pathogenic bacteria are composed of diverse and complex carbohydrate structures, some of which are known virulence factors. Indeed, different B. cereus peptidoglycans and glycoproteins were isolated, and some were reported to play a role in spore formation and infection (3-5). It is also clear, however, that among those different types of Bacillus glycans, many are not yet fully characterized. Regardless, compared with the limited knowledge of these glycan structures, the pathways leading to their biosyntheses are still elusive. To identify such metabolic pathways, we initially decided to look for putative genes encoding enzymes involved in the synthesis of glycan precursors (i.e. nucleotide-sugars).Different UDP-GlcA 2 decarboxylases with distinct functions exist in both eukaryotes...

Discovery of a Unique Extracellular Polysaccharide in Members of the Pathogenic Bacillus That Can Co-form with Spores

“…2D). The retention time and m/z values are identical with those found for alditol acetate derivatives of a XylNAc standard derived from UDP-XylNAc (27). Hence the major neutral sugar in pzX is XylNAc.…”

“…Hence the major neutral sugar in pzX is XylNAc. Because previous works showed that UDP-XylNAc is made by the enzymatic actions of Bc0487 and Bc0488 (27), we addressed whether the putative glycosyltransferase (Bc0486) that co-resides in the operon facilitates pzX formation. To this end, we generated Bc0486 mutant (⌬bc0486) and complemented strains.…”

“…ing the finding of genes and the enzymes involved in UDP-GlcNAcA and UDP-XylNAc synthesis (27), the identification of glycan(s) consisting of these unusual amino sugars was unsuccessful when Bacillus cells were growing under typical laboratory conditions. We therefore decided to grow Bacillus in different growth media to find conditions that stimulate transcription of genes in the XNAC operon and promote the synthesis of a XylNAc-containing molecule.…”

“…In 2010, Gu et al (27) identified two enzymes (Fig. 1, A and B) and their corresponding genes in Bacillus food pathogen and showed their involvement in the sequential conversion of UDP-N-acetylglucosamine to UDP-N-acetylglucosaminuronic acid and then to UDP-N-acetylxylosamine.…”

“…A, organization and conserved synteny of genes within the XNAC operon. B, biochemical pathways for the conversion of UDP-GlcNAc to UDP-XylNAc by UGlcNAcDH and UXylNAcS (27) within the XNAC operon are illustrated. A phylogenetic tree of UXylNAcS (C) depicts that XNAC operon is only present in members of the B. cereus sensu lato group.…”