The chemical structure of the lipid A components of smooth‐type lipopolysaccharides isolated from... more The chemical structure of the lipid A components of smooth‐type lipopolysaccharides isolated from the type strains of strictly anaerobic beer‐spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were analyzed. The hydrophilic backbone of lipid A was shown, by controlled degradation of lipopolysaccharide combined with chemical assays and 31P‐NMR spectroscopy, to consist of the common β1‐6‐linked disaccharide of pyranosidic 2‐deoxy‐glucosamine (GlcN), phosphorylated at the glycosidic position and at position 4′. In de‐O ‐acylated lipopolysaccharide, the latter phosphate was shown to be quantitatively substituted with 4‐amino‐4‐deoxyarabinose, whereas the glycosidically linked phosphate was present as a monoester. Laser‐desorption mass spectrometry of free dephosphorylated lipid A revealed that the distal (non‐reducing) GlcN was substituted at positions 2′ and 3′ with (R)‐3‐(undecanoyloxy)tridecanoic acid, whereas the reducing GlcN carried two unsubstituted (R)‐3‐hydroxytetradecanoic acids at positions 2 and 3. The lipid A of both Pectinatus species were thus of the asymmetric hexaacyl type. The linkage of lipid A to polysaccharide in the lipopolysaccharide was relatively resistant to acid‐catalyzed hydrolysis, enabling the preparation of a dephosphorylated and deacylated saccharide backbone. Methylation analysis of the backbone revealed that position 6′ of the distal GlcN of lipid A was the attachment site of the polysaccharide. Despite the quantitative substitution of the lipid A 4′‐phosphate by 4‐amino‐4‐deoxyarabinose, which theoretically should render the bacteria resistant to polymyxin, P. cerevisiiphilus was shown to be susceptible to this antibiotic. P. cerevisiiphilus was, however, also susceptibile to vancomycin and bacitracin, indicating that the outer membrane of this bacterium does not act as an effective permeability barrier.
The polyamines putrescine, cadaverine, spermidine, and spermine and the corresponding mono-N-acet... more The polyamines putrescine, cadaverine, spermidine, and spermine and the corresponding mono-N-acetylpolyamines can be separated as their dimethylaminoazobenzenesulfonyl derivatives in a single analysis in less than 22 min. The method employs reversed-phase high-performance liquid chromatography (Spherisorb S5 ODS2 column) with an acetonitrile/acetate buffer gradient elution system and detection in the visible (436 nm) region. The detection limit for a single dimethylaminoazobenzenesulfonylpolyamine is less than 2 pmol.
International Journal of Food Microbiology, Sep 1, 2000
The effect of nisin pretreatment on organic acid-induced permeability increase in strains of Esch... more The effect of nisin pretreatment on organic acid-induced permeability increase in strains of Escherichia coli, Pseudomonas aeruginosa, P. marginalis, and Salmonella enterica sv. Typhimurium was investigated, using assays based on the uptake of a fluorescent dye 1-N-phenylnaphthylamine (NPN) and on the bacterial susceptibility to detergent-induced bacteriolysis. The outer membrane of bacteria which had been pretreated with nisin was shown to be less stable against 1 mM EDTA, as indicated by their significantly higher NPN uptake levels as compared to untreated bacteria. Upon challenge with a tenfold lower concentration of EDTA (0.1 mM) some nisin-treated strains (Typhimurium, P. marginalis) exhibited, however, NPN uptake levels which were lower than those seen in control bacteria, suggesting that nisin had stabilized their outer membrane. Nisin pretreatment also decreased the NPN uptake induced by citric or lactic acid or both in E. coli, P. marginalis, and Typhimurium, whereas in P. aeruginosa the pretreatment resulted in increased NPN uptake in response to citric and lactic acid. These results suggest that, with the exception of P. aeruginosa, nisin could protect bacteria from the outer membrane-disrupting effect caused by the acids. P. aeruginosa was, however, shown to be protected against bacteriolysis induced by the detergents sodium dodecylsulfate and Triton X-100. With a pair of isogenic mutants of Typhimurium differing in their cell surface charge it was shown that the NPN uptake response to I mM EDTA of the abnormally cationic strain was not significantly affected by nisin, whereas in the normal anionic strain nisin strongly strengthened the uptake. Our hypothesis based on these findings is that the normally anionic cell surface of Gram-negative bacteria has a tendency to bind the cationic nisin. The binding of nisin to the surface does not proceed to the cytoplasmic membrane, but in the outer membrane the bound nisin actually stabilizes its structure through electrostatic interactions. With the exception of EDTA, the organic acids at pH 4 did not cause leakage of cell contents from Typhimurium, indicating that these acids do not permeabilize the outer membrane to an extent required for cytoplasmic pore formation by nisin.
The chemical structure of the lipid A components of smooth‐type lipopolysaccharides isolated from... more The chemical structure of the lipid A components of smooth‐type lipopolysaccharides isolated from the type strains of strictly anaerobic beer‐spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were analyzed. The hydrophilic backbone of lipid A was shown, by controlled degradation of lipopolysaccharide combined with chemical assays and 31P‐NMR spectroscopy, to consist of the common β1‐6‐linked disaccharide of pyranosidic 2‐deoxy‐glucosamine (GlcN), phosphorylated at the glycosidic position and at position 4′. In de‐O ‐acylated lipopolysaccharide, the latter phosphate was shown to be quantitatively substituted with 4‐amino‐4‐deoxyarabinose, whereas the glycosidically linked phosphate was present as a monoester. Laser‐desorption mass spectrometry of free dephosphorylated lipid A revealed that the distal (non‐reducing) GlcN was substituted at positions 2′ and 3′ with (R)‐3‐(undecanoyloxy)tridecanoic acid, whereas the reducing GlcN carried two unsubstituted (R)‐3‐hydroxytetradecanoic acids at positions 2 and 3. The lipid A of both Pectinatus species were thus of the asymmetric hexaacyl type. The linkage of lipid A to polysaccharide in the lipopolysaccharide was relatively resistant to acid‐catalyzed hydrolysis, enabling the preparation of a dephosphorylated and deacylated saccharide backbone. Methylation analysis of the backbone revealed that position 6′ of the distal GlcN of lipid A was the attachment site of the polysaccharide. Despite the quantitative substitution of the lipid A 4′‐phosphate by 4‐amino‐4‐deoxyarabinose, which theoretically should render the bacteria resistant to polymyxin, P. cerevisiiphilus was shown to be susceptible to this antibiotic. P. cerevisiiphilus was, however, also susceptibile to vancomycin and bacitracin, indicating that the outer membrane of this bacterium does not act as an effective permeability barrier.
The polyamines putrescine, cadaverine, spermidine, and spermine and the corresponding mono-N-acet... more The polyamines putrescine, cadaverine, spermidine, and spermine and the corresponding mono-N-acetylpolyamines can be separated as their dimethylaminoazobenzenesulfonyl derivatives in a single analysis in less than 22 min. The method employs reversed-phase high-performance liquid chromatography (Spherisorb S5 ODS2 column) with an acetonitrile/acetate buffer gradient elution system and detection in the visible (436 nm) region. The detection limit for a single dimethylaminoazobenzenesulfonylpolyamine is less than 2 pmol.
International Journal of Food Microbiology, Sep 1, 2000
The effect of nisin pretreatment on organic acid-induced permeability increase in strains of Esch... more The effect of nisin pretreatment on organic acid-induced permeability increase in strains of Escherichia coli, Pseudomonas aeruginosa, P. marginalis, and Salmonella enterica sv. Typhimurium was investigated, using assays based on the uptake of a fluorescent dye 1-N-phenylnaphthylamine (NPN) and on the bacterial susceptibility to detergent-induced bacteriolysis. The outer membrane of bacteria which had been pretreated with nisin was shown to be less stable against 1 mM EDTA, as indicated by their significantly higher NPN uptake levels as compared to untreated bacteria. Upon challenge with a tenfold lower concentration of EDTA (0.1 mM) some nisin-treated strains (Typhimurium, P. marginalis) exhibited, however, NPN uptake levels which were lower than those seen in control bacteria, suggesting that nisin had stabilized their outer membrane. Nisin pretreatment also decreased the NPN uptake induced by citric or lactic acid or both in E. coli, P. marginalis, and Typhimurium, whereas in P. aeruginosa the pretreatment resulted in increased NPN uptake in response to citric and lactic acid. These results suggest that, with the exception of P. aeruginosa, nisin could protect bacteria from the outer membrane-disrupting effect caused by the acids. P. aeruginosa was, however, shown to be protected against bacteriolysis induced by the detergents sodium dodecylsulfate and Triton X-100. With a pair of isogenic mutants of Typhimurium differing in their cell surface charge it was shown that the NPN uptake response to I mM EDTA of the abnormally cationic strain was not significantly affected by nisin, whereas in the normal anionic strain nisin strongly strengthened the uptake. Our hypothesis based on these findings is that the normally anionic cell surface of Gram-negative bacteria has a tendency to bind the cationic nisin. The binding of nisin to the surface does not proceed to the cytoplasmic membrane, but in the outer membrane the bound nisin actually stabilizes its structure through electrostatic interactions. With the exception of EDTA, the organic acids at pH 4 did not cause leakage of cell contents from Typhimurium, indicating that these acids do not permeabilize the outer membrane to an extent required for cytoplasmic pore formation by nisin.
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Papers by Ilkka Helander