Components of the wall and capsule of Bacillus megaterium NCIB 7581

Bishop, Alistair H.; Douglas, C. W. I.; Dorr, Patrick; White, Peter J

doi:10.1099/00221287-139-11-2723

Cited by 2 publications

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References 25 publications

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“…An acidic accessory polymer containing glucose and glucosamine was recognized in walls of Bacillus megaterium NCIB 758 1 (preceding paper : Bishop et al, 1993). The present paper describes a further study of this polymer which has led to the identification of N-lactyl-3-amino-3,6-dideoxyhexose as a major component.…”

Section: Introductionmentioning

confidence: 92%

“…Bacillus megaterium NCIB 758 1 and the double mutant GW-1 which requires diaminopimelate plus lysine for growth are described by Bishop et al (1993).…”

Section: Methodsmentioning

confidence: 99%

“…1 paper. Solvents A and B were as described by Bishop et al (1993); solvents C, D and E were from Jann et al (1967); solvent G was toluene/butan-l-ol/water (1 : 9:2, by vol., upper phase: Renkonen & Schindler, 1956). The two solvents for the method of Howe (1960) were also used.…”

Section: Separation Techniques (A)mentioning

confidence: 99%

“…0.88) in 3 1 water. To reveal spots after chromatography or electrophoresis the following reagents were used: ninhydrin for amino compounds; p-anisidine for sugars (both used as described by Bishop et al, 1993); periodate/benzidine reagents (Gordon et al, 1956) for periodate-sensitive compounds ; bromophenol blue (40 mg in 95 YO, v/v, aqueous ethanol, adjusted to pH 6.5) for acids, which appeared as yellow spots against a blue background. ( d ) HPLC.…”

Section: Separation Techniques (A)mentioning

confidence: 99%

“…Isomers of lactate were measured with L-and D-lactate dehydrogenases, as described by Gutmann & Wahlfeld (1974) and Gawehn & Bergmeyer (1974). Acetate was measured as described by Bishop et al (1993).…”

Section: Separation Techniques (A)mentioning

confidence: 99%

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Composition of an unusual accessory polymer containing N-lactyl-3-amino-3,6-dideoxyhexose from walls of Bacillus megaterium NCIB 7581

Bishop

White

1993

Journal of General Microbiology

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A polysaccharide of molecular mass at least 300000 Da was isolated from walls of BaciZZus megateriurn NCIB 7581. Three sugars, N-lactyl-3-~0-3,6dideoxyhexose, N-acetylglucosamine and glucose were present in equimolar proportions, and accounted for 90 % of the carbon of the polysaccharide. The remainder of the polymer was an unidentified acidic molecule, with a hydrophobic nature. IntroductionAn acidic accessory polymer containing glucose and glucosamine was recognized in walls of Bacillus megaterium NCIB 758 1 (preceding paper : Bishop et al., 1993). The present paper describes a further study of this polymer which has led to the identification of N-lactyl-3-amino-3,6-dideoxyhexose as a major component. MethodsOrganisms. Bacillus megaterium NCIB 758 1 and the double mutant GW-1 which requires diaminopimelate plus lysine for growth are described by Bishop et al. (1993).Growth of bacteria, isolation of walls and of the acidic polymer. All of these procedures were as described before (Bishop et al., 1993).Tests with lectins. The lysozyme-extracted polymer was tested against concanavalin A Type V (from Sigma) by the agar diffusion method of Goldstein & So (1965) with Type I1 glycogen (Sigma) as positive control, and against Solanum tuberosum lectin with a positive control of peptidoglycan (B. megaterium) from which the accessory polymer had been removed by extraction with HC1.Hydrolysis of the polymer. Routinely the polymer (irrespective of the method of isolation) was hydrolysed as a solution (3 mg ml-' or less) in 2 M-HCl in a sealed tube for 2 h in boiling water. When diaminopimelate was to be measured colorirnetrically, hydrolysis was at 105 "C in 6 M-HCl for 18 h. Hydrolysates were dried repeatedly in a desiccator over silica gel and solid NaOH, or else were freeze-dried. Hydrolysis for only 20 min in 1 M-HC~ in boiling water liberated N-lactylaminodideoxyhexose optimally. Isolation of oligosaccharides from partial hydrolysates of the polymer.Lysozyme-extracted polymer (20 mg) was kept at 100 "C in 0 1 M-HCl (15 ml) for 2 h. The hydrolysate was rotary evaporated, taken up in water (1-5 ml) and eluted with water through a column of Bio-Gel P2. Samples from fractions were assayed, and two discrete peaks of anthrone-positive material were found to emerge between the void volume and the elution volume of monosaccharides. These two impure oligosaccharide isolates were freeze-dried, and each was separately recovered from Dowex-50 with O~M -H C~, then eluted once more through the Bio-Gel P2 column.Treatment of polymer with HF. Lysozyme-extracted polymer (6 mg) or walls of Lactobacillus plantarum (6 rng) were separately added to 300 pl HF (50 %, w/v) at 0 O C in a plastic tube. The mixtures were left at 0 "C for 1 h with occasional shaking, then the acid was carefully neutralized with 10 M-NaOH while the temperature was kept at 0 "C. Fine adjustment to pH 7 was done with 1 M-NaOH and 1 M-HCl, using indicator paper. The wall suspension was centrifuged and the supernatant liquid was kept. Both neutralized solutions were...

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Section: Introductionmentioning

confidence: 92%

“…Bacillus megaterium NCIB 758 1 and the double mutant GW-1 which requires diaminopimelate plus lysine for growth are described by Bishop et al (1993).…”

Section: Methodsmentioning

confidence: 99%