Abstract
Surface glycan switching is often observed when micro-organisms transition between different biotic and abiotic niches, including biofilms, although the advantages of this switching to the organism are not well understood. Bacillus cereus grown in a biofilm-inducing medium has been shown to synthesize an unusual cell wall polysaccharide composed of the repeating subunit →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(β1→, where galactose is linked to the hydroxyglutarate moiety of FucNAc-4-amido-(2)-hydroxyglutarate. The molecular mechanism involved in attaching 2-hydroxyglutarate to 4-amino-FucNAc has not been determined. Here, we show two genes in B. cereus ATCC 14579 encoding enzymes involved in the synthesis of UDP-FucNAc-4-amido-(2)-oxoglutarate (UDP-Yelosamine), a modified UDP-sugar not previously reported to exist. Using mass spectrometry and real time NMR spectroscopy, we show that Bc5273 encodes a C4″-aminotransferase (herein referred to as Pat) that, in the presence of pyridoxal phosphate, transfers the primary amino group of l-Glu to C-4″ of UDP-4-keto-6-deoxy-d-GlcNAc to form UDP-4-amino-FucNAc and 2-oxoglutarate. Pat also converts 4-keto-xylose, 4-keto-glucose, and 4-keto-2-acetamido-altrose to their corresponding UDP-4-amino-sugars. Bc5272 encodes a carboxylate-amine ligase (herein referred as Pyl) that, in the presence of ATP and Mg(II), adds 2-oxoglutarate to the 4-amino moiety of UDP-4-amino-FucNAc to form UDP-Yelosamine and ADP. Pyl is also able to ligate 2-oxoglutarate to other 4-amino-sugar derivatives to form UDP-Yelose, UDP-Solosamine, and UDP-Aravonose. Characterizing the metabolic pathways involved in the formation of modified nucleotide sugars provides a basis for understanding some of the mechanisms used by bacteria to modify or alter their cell surface polysaccharides in response to changing growth and environmental challenges.
Highlights
Activated sugars required to form rare oligosaccharide subunits of Bacillus cereus biofilm-polysaccharide are not known
Bacillus cereus grown in a biofilm-inducing medium has been shown to synthesize an unusual cell wall polysaccharide composed of the repeating subunit 36)Gal(␣1–2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(␣1– 6)GlcNAc(13, where galactose is linked to the hydroxyglutarate moiety of FucNAc-4-amido-(2)-hydroxyglutarate
Using mass spectrometry and real time NMR spectroscopy, we show that Bc5273 encodes a C4؆-aminotransferase that, in the presence of pyridoxal phosphate, transfers the primary amino group of L-Glu to C-4؆ of UDP-4-keto-6-deoxy-D-GlcNAc to form UDP-4-amino-FucNAc and 2-oxoglutarate
Summary
Activated sugars required to form rare oligosaccharide subunits of Bacillus cereus biofilm-polysaccharide are not known. Bacillus cereus grown in a biofilm-inducing medium has been shown to synthesize an unusual cell wall polysaccharide composed of the repeating subunit 36)Gal(␣1–2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(␣1– 6)GlcNAc(13, where galactose is linked to the hydroxyglutarate moiety of FucNAc-4-amido-(2)-hydroxyglutarate. We show two genes in B. cereus ATCC 14579 encoding enzymes involved in the synthesis of UDP-FucNAc-4-amido-(2)-oxoglutarate (UDP-Yelosamine), a modified UDP-sugar not previously reported to exist. Candela et al (6) described a second SCWP with a repeating sequence of 36)Gal(␣1–2)(2-Rhydroxyglutar-5-ylamido)Fuc2NAc4N(␣1– 6)GlcNAc(13 This SCWP was detected in the planktonic phase and in late stage biofilms when the bacterium was grown in a selective nutrient medium rather than a nutrient-rich medium (6). The first enzyme is a C4Љ-aminotransferase (Pat), which catalyzes the transfer of the primary amino group of L-Glu to C-4 of UDP-4-keto-6-deoxy-D-GlcNAc to form UDP-4-amino-FucNAc and 2-oxoglutarate (2OG). Amino acid sequence homology suggests that this ATP-dependent enzyme is a member of the ATP-Grasp family of proteins (8, 9)
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