Abstract
CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-d-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-D-GlcNAc-5,6-ene. Pen contains strongly bound NADP(+) and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-D-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-L-AltNAc. Pal is NAD(+)-dependent and has distinct UDP-6-deoxy-d-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-L-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4″-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.
Highlights
CMP-pseudaminic acid formation and role in Bacillus thuringiensis flagellin glycosylation are unknown
CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection
CMP-Pseudaminic Acid Operon in B. thuringiensis Consists of Seven Genes—We first performed a BLAST search using amino acid sequences of known bacterial enzymes involved in CMP-pseudaminic acid (CMP-proteins because sugar (Pse)) formation to identify B. thuringiensis genes that may have a role in CMP-Pse formation
Summary
CMP-pseudaminic acid formation and role in Bacillus thuringiensis flagellin glycosylation are unknown. We identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. Interest in the function of Pse in Gram-negative bacteria led to the identification of a biosynthetic pathway, named pse, where CMP-pseudaminic acid (CMP-Pse) is formed from UDP-GlcNAc [27,28,29]. This pathway involves six enzymes (Fig. 1A). The identification of Pen and Pal and the CMP-Pse biosynthetic pathway in B. thuringiensis provides a basis for determining the biological role of Pse in flagellin glycosylation in Gram-positive bacterium
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