Purification and Characterization of Phosphonoglycans from Glycomyces sp. Strain NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338

• Published in: Journal of bacteriology Vol. 196; no. 9; pp. 1768 - 1779
• Main Authors: Yu, Xiaomin, Price, Neil P. J, Evans, Bradley S, Metcalf, William W
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.05.2014
• Subjects: acid hydrolysis; Actinomycetales - chemistry; Actinomycetales - enzymology; Actinomycetales - genetics; Actinomycetales - metabolism; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; biosynthesis; Carbohydrate Sequence; exopolysaccharides; galactose; Genomes; Glucose; Glycomyces; isotope labeling; Magnetic Resonance Spectroscopy; Mass spectrometry; methanol; Molecular Sequence Data; Molecular Weight; Molecules; multigene family; nuclear magnetic resonance spectroscopy; nucleotide sequences; Organophosphonates - metabolism; phosphonates; phosphorous acid; Phosphotransferases (Phosphomutases) - genetics; Phosphotransferases (Phosphomutases) - metabolism; Polysaccharides, Bacterial - chemistry; Polysaccharides, Bacterial - isolation & purification; Polysaccharides, Bacterial - metabolism; S-adenosylmethionine; screening; solvents; ultrafiltration; xylose
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/jb.00036-14

Two related actinomycetes, Glycomyces sp. strain NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338, were identified as potential phosphonic acid producers by screening for the gene encoding phosphoenolpyruvate (PEP) mutase, which is required for the biosynthesis of most phosphonates. Using a variety of analytical techniques, both strains were subsequently shown to produce phosphonate-containing exopolysaccharides (EPS), also known as phosphonoglycans. The phosphonoglycans were purified by sequential organic solvent extractions, methanol precipitation, and ultrafiltration. The EPS from the Glycomyces strain has a mass of 40 to 50 kDa and is composed of galactose, xylose, and five distinct partially O-methylated galactose residues. Per-deutero-methylation analysis indicated that galactosyl residues in the polysaccharide backbone are 3,4-linked Gal, 2,4-linked 3-MeGal, 2,3-linked Gal, 3,6-linked 2-MeGal, and 4,6-linked 2,3-diMeGal. The EPS from the Stackebrandtia strain is comprised of glucose, galactose, xylose, and four partially O-methylated galactose residues. Isotopic labeling indicated that the O-methyl groups in the Stackebrandtia phosphonoglycan arise from S-adenosylmethionine. The phosphonate moiety in both phosphonoglycans was shown to be 2-hydroxyethylphosphonate (2-HEP) by 31P nuclear magnetic resonance (NMR) and mass spectrometry following strong acid hydrolysis of the purified molecules. Partial acid hydrolysis of the purified EPS from Glycomyces yielded 2-HEP in ester linkage to the O-5 or O-6 position of a hexose and a 2-HEP mono(2,3-dihydroxypropyl)ester. Partial acid hydrolysis of Stackebrandtia EPS also revealed the presence of 2-HEP mono(2,3-dihydroxypropyl)ester. Examination of the genome sequences of the two strains revealed similar pepM-containing gene clusters that are likely to be required for phosphonoglycan synthesis.