The purification and chemical composition of the lipopolysaccharide of Pseudomonas alcaligenes.
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ABSTRACT:
SUBMITTER: Key BA
PROVIDER: S-EPMC1179637 | biostudies-other | 1970 Dec
REPOSITORIES: biostudies-other
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Project description:1. Qualitative and quantitative analytical results for the lipopolysaccharide from acetone-dried cells of Pseudomonas aeruginosa (N.C.T.C. 1999) are presented and possible contamination of the material with nucleic acid was further examined. 2. Additional sugars detected (only in large-scale hydrolysates) were mannose and arabinose; traces of spermidine and putrescine were also found. 3. The heptose component is l-glycero-d-mannoheptose. 4. The thiobarbituric acid-positive component is a 3-deoxy-2-octulonic acid, of which only 35-40% links lipid A to the polysaccharide. This linkage is not broken by hydrolysis with acetic acid up to 0.08m. 5. Liberation of lipid A required hydrolysis with 0.1m-hydrochloric acid, which substantially degraded the polysaccharide moiety. 6. Fractions obtained from the degraded polysaccharide by high-voltage electrophoresis were examined; in these, the alanine/galactosamine molar ratio is approx. 1. 7. Hydrazinolysis of whole lipopolysaccharide showed that at least 40% of the alanine is in amide linkage, possibly with galactosamine. 8. Lipid A, solubilized by alkaline methanolysis was fractionated; most of the phosphorus of the higher-molecular-weight fractions was released as P(i) by a phosphomonoesterase. 9. Hydrazinolysis of lipid A destroyed approx. 80% of the glucosamine, and glycosidically linked glucosamine oligosaccharides could not be isolated.
| S-EPMC1178387 | biostudies-other
Project description:Studies of the lipopolysaccharide of Pseudomonas alcaligenes strain BR 1/2 were extended to the polysaccharide moiety. The crude polysaccharide, obtained by mild acid hydrolysis of the lipopolysaccharide, was fractionated by gel filtration. The major fraction was the phosphorylated polysaccharide, for which the approximate proportions of residues were; glucose (2), rhamnose (0.7), heptose (2-3), galactosamine (1), alanine (1), 3-deoxy-2-octulonic acid (1), phosphorus (5-6). The heptose was l-glycero-d-manno-heptose. The minor fractions from gel filtration contained free 3-deoxy-2-octulonic acid, P(i) and PP(i). The purified polysaccharide was studied by periodate oxidation, methylation analysis, partial hydrolysis, and dephosphorylation. All the rhamnose and part of the glucose and heptose occur as non-reducing terminal residues. Other glucose residues are 3-substituted, and most heptose residues are esterified with condensed phosphate residues, possibly in the C-4 position. Free heptose and a heptosylglucose were isolated from a partial hydrolysate of the polysaccharide. The location of galactosamine in the polysaccharide was not established, but either the C-3 or C-4 position appears to be substituted and a linkage to alanine was indicated. In its composition, the polysaccharide from Ps. alcaligenes resembles core polysaccharides from other pseudomonads: no possible side-chain polysaccharide was detected.
| S-EPMC1166327 | biostudies-other
Project description:Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer's patches. We previously reported that an A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4'-phosphates. Hexaacylated A. faecalis lipid A showed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit a discrete interleukin-6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate.
| S-EPMC8252424 | biostudies-literature
Project description:1. Lipopolysaccharide was isolated from both cell walls and acetone-dried whole cells of Pseudomonas aeruginosa (N.C.T.C. 1999). 2. Closely similar products are obtained, although that from whole cells cannot be completely freed from small amounts (2-7%) of residual nucleic acids. 3. The lipid moiety (23-33%) has a similar amino sugar backbone to that of lipids of enterobacterial lipopolysaccharides, but contains different hydroxy acids (2- and 3-hydroxydodecanoic acid and 3-hydroxydecanoic acid). 3-Hydroxytetradecanoic acid is absent, and 3-hydroxydodecanoic acid is the main N-acylating acid. No clear evidence permitting a distinction between the possibilities that phosphodiester or glycosidic linkages exist between the glucosamine residues was obtained. 4. Identifiable sugars (glucose, rhamnose, 3-deoxy-2-octulonic acid and heptose) account for less than 20% of the lipopolysaccharide, and alanine, galactosamine and fucosamine are apparently components of the polysaccharide moiety. 5. The polysaccharide moiety is unusual in that it is not readily obtained from the lipopolysaccharide by treatment with dilute acetic acid, which does, however, solubilize much of the phosphorus of the lipopolysaccharide. 6. The ;polysaccharide' fraction (approx. 21%) obtained by treatment with dilute acetic acid contains only a small proportion of the total polysaccharide components, and in one case only 45% of the fraction was accountable for in terms of identifiable components. 7. Evidence suggests that unidentified nitrogenous components are concentrated in the residual material after removal of both the lipid and the ;polysaccharide' fraction from the lipopolysaccharide.
| S-EPMC1184843 | biostudies-other
Project description:Pseudomonas alcaligenes DIP1 produces an extracellular cyanophycinase (CphEal). The corresponding gene (cphEal) was identified from subclones of a genomic DNA gene library by heterologously expressing the functionally active enzyme in Escherichia coli. The nucleotide sequence of the gene (1260 base pairs) was determined indicating a theoretical mass of 43.6 kDa (mature CphEal) plus a leader peptide of 2,6 kDa which corresponds well to the apparent molecular mass of 45 kDa as revealed by SDS-PAGE. The enzyme exhibited a high sequence identity of 91% with the extracellular cyanophycinase from P. anguilliseptica strain BI and carried an N-terminal Sec secretion signal peptide. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the serine motif GXSXG plus a histidine and a glutamate residue, suggesting a catalytic mechanism similar to serine-type proteases. The cyanophycinase (CphEal) was heterologously produced in two different E. coli strains (Top10 and BL21(DE3)) from two plasmid vectors (pBBR1MCS-4 and pET-23a(+)). The signal peptide of CphEal was cleaved in E. coli, suggesting active export of the protein at least to the periplasm. Substantial enzyme activity was also present in the culture supernatants. The extracellular cyanophycinase activities in E. coli were higher than activities in the wild type P. alcaligenes DIP1 in complex LB medium. Highest extracellular enzyme production was achieved with E. coli BL21(DE3) expressing CphEal from pBBR1MCS-4. Using M9 minimal medium was less effective, but the relatively low cost of mineral salt media makes these results important for the industrial-scale production of dipeptides from cyanophycin.
| S-EPMC3235067 | biostudies-literature
Project description:This study is to investigate the use of microbial remediation technology for degradation of dexamethasone in polluted water. A strain of Pseudomonas Alcaligenes with the ability of dexamethasone degradation was isolated from hospital polluted water. This strain was further acclimated into a bacterial strain that could highly degrade dexamethasone. Domesticated bacterial proteins were separated by osmotic shock method and were analyzed using SDS-PAGE. Enzyme activity of dexamethasone degradation was detected by high performance liquid chromatography. Protein bands with different molecular weight were found in all regions of the bacteria and a band with molecular weight of about 100 kDa was most obvious. In intracellular and periplasmic liquid, there was a band with molecular weight of about 41 kDa. Enzyme activity mainly existed in intracellular liquid. The 41 kDa protease was purified using ammonium sulfate precipitation, DEAE-52 ion exchange column and Sephadex G-100 column. Dexamethasone and dexamethasone sodium phosphate degrading rates of the purified enzyme were 36% and 95%, respectively. The 100 kDa protein had a 19% coverage rate to TonB receptor dependent protein, with 11 peptides matching. The 41 kDa protein had a 56% coverage rate to isovaleryl coenzyme A dehydrogenase, with 5 peptides matching. The 41 kDa protein had good degradation between the temperature of 25-40°C and PH value of 6.5-8.5. The enzyme kinetics equation was Ct = C0 e(-0.1769t), in accordance with the first-order kinetic equation. This study laid the foundation for further preparation of bioremediation agents for clearance of dexamethasone pollution in water.
| S-EPMC4565275 | biostudies-literature
Project description:1. EDTA in borate buffer has a marked bactericidal effect on Pseudomonas alcaligenes, which is more sensitive than Pseudomonas aeruginosa. The bactericidal effect is accompanied by solubilization of lipopolysaccharide and release of intracellular solutes. These effects are more pronounced at pH9.2 than 7.1. 2. Cell walls of P. alcaligenes were prepared and from them were obtained the readily extracted lipids and the fractions given by treatment with aqueous phenol. 3. The cell walls and the above components were analysed and results are compared with those for P. aeruginosa. 4. Lipopolysaccharide obtained by treatment of cell walls with aqueous phenol is contaminated with glycosaminopeptide to a variable extent. 5. The lipopolysaccharide contains less neutral sugar but more phosphorus than the lipopolysaccharide of P. aeruginosa; fucosamine is not a component of the lipopolysaccharide of P. alcaligenes.
| S-EPMC1179024 | biostudies-other
Project description:Pseudomonas alcaligenes Raw sequence reads
| PRJNA1032269 | ENA
Project description:Inorganic arsenic (As) is highly toxic and ubiquitous in the environment. Inorganic As can be transformed by microbial methylation, which constitutes an important part of the As biogeochemical cycle. In this study, we investigated As biotransformation by Pseudomonas alcaligenes NBRC14159. P. alcaligenes was able to methylate arsenite [As(III)] rapidly to dimethylarsenate and small amounts of trimethylarsenic oxide. An arsenite S-adenosylmethionine methyltransferase, PaArsM, was identified and functionally characterized. PaArsM shares low similarities with other reported ArsM enzymes (<55%). When P. alcaligenes arsM gene (PaarsM) was disrupted, the mutant lost As methylation ability and became more sensitive to As(III). PaarsM was expressed in the absence of As(III) and the expression was further enhanced by As(III) exposure. Heterologous expression of PaarsM in an As-hypersensitive strain of Escherichia coli conferred As(III) resistance. Purified PaArsM protein methylated As(III) to dimethylarsenate as the main product in the medium and also produced dimethylarsine and trimethylarsine gases. We propose that PaArsM plays a role in As methylation and detoxification of As(III) and could be exploited in bioremediation of As-contaminated environments.
| S-EPMC4375323 | biostudies-literature