Ribitol-containing lipopolysaccharides from Proteus mirabilis and their serological relationship.

Ribitol phosphate was recently identified as a constituent of lipopolysaccharides obtained from 'proteus mirabilis strain D52 giving 1:4-anhydroribitol during acid hydrolysis (Gmeiner, 1975). Two other Proteus mirabilis strains belonging to serogroups O16 and O33 were shown previously to contain an unknown compoound X as lipopolysaccharide constituent (Kotelko et al., 1975). In this report the identification of compound X as 1:4-anhydroribotol by gas-liquid chromatography, mass spectrometry and mass fragmentography is described. Serological investigations using passive hemagglutination, hemagglutination inhbition and semi-quantitative precipitin reactions indicate strongly that ribitol plays a role in the serological specificity of the respective lipopolysaccharides.     

The effect of environmental iron concentration on R-plasmid RP1-mediated changes in the lipopolysaccharide and fatty acid composition of Proteus mirabilis

Abstract Rhizobium meliloti JJ-1 grown in a medium rich in manganese elaborated an exopolysaccharide (EPS) that showed marked differences from that produced in the same medium deficient in added manganese. Glucose, galactose and mannose appeared to be the primary carbohydrate constituents of the EPS isolated from the spent fluid of the control culture, while that from the manganese-enriched broth was comprised mainly of glucose and galactose. Examination of these biopolymers by ¹³C-NMR spectroscopy also revealed substantial differences in their non-carbohydrate moieties.     

Cerulenin-induced changes in the lipopolysaccharide content and phospholipid composition of Proteus mirabilis.

Inhibition of Proteus mirabilis growth by cerulenin, a specific inhibitor of fatty acid biosynthesis, was reversed by exogenously supplied fatty acid mixtures containing oleic acid and palmitic or pentadecanoic acids. The growth rate of the cells treated with cerulenin in the presence of the fatty acid mixtures was slower, however, than that of untreated cells, and their lipopolysaccharide content was decreased by 30-50%, resulting in an increased sensitivity of the organisms to rifamycin and vancomycin. Polyacrylamide gel electrophoresis of the lipopolysaccharide fraction from cerulenin-treated cells revealed that of the two P. mirabilis lipopolysaccharide types, the relative amount of the higher molecular weight lipopolysaccharide was reduced from 50% to 30% of the total lipopolysaccharide. Fatty acid analysis of the phospholipid and lipopolysaccharide fractions from cells grown with cerulenin, pentadecanoate, and oleate revealed that over 60% of the native even-numbered fatty acids of the phospholipid fraction was substituted by the odd-numbered fatty acid, while no incorporation of either the pentadecanoate or oleate could be demonstrated in the lipid A moiety of the lipopolysaccharide. The only change in the lipid A observed was an increase in the content of 3-hydroxymyristic acid accompanied by a decrease in the nonhydroxylated fatty acids, supporting the highly conserved nature of this molecule.     

Human fetal and adult spleen phospholipids and their fatty acid composition

Total phospholipid contents and the individual phospholipid components of human adult and fetal spleens from 17--18 and 23--24 week's pregnancies composition of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, diphosphatidylglycerol and phosphatidic acid studied in human adult, 17--18 and 23--24 week fetal spleens.     

The role of defensin fragment in the regulation of fatty acid composition of membrane phospholipids in epithelial-like cells

It is shown that a tetrapeptide fragment of defensin does not alter the phospholipid composition in the membranes of CHO-K1 cells but regulates the fatty acid composition of phosphatidylcholine, phosphatidylethanolamine (PEA), phosphatidylserine (PS), and phosphatidylinositol (PI). Incubation of the cells in the presence of this tetrapeptide resulted in modification of unsaturated fatty acid composition in the studied phospholipids. The content of monoenoic (mainly C18 : 1ω9) and/or dienoic (C18 : 2ω6) fatty acids increased, while the level of polyenoic fatty acids decreased. It was found that in the polyenoic fatty acid group of the PEA, PS and PI molecules, the ω3-/ω6-acid ratio decreased mainly due to the lower content of long-chain ω3-acids with 20 and/or 22 carbonic atoms. The possible role of this peptide in inhibition of the activity of Δ6- and Δ5-desaturases involved in the synthesis of long-chain polyenoic fatty acids, the quantitative alteration of which in phospholipids influences physicochemical parameters in cell membranes, is discussed.     

Thermal regulation of fatty acid synthetase from Brevibacterium ammoniagenes.

The fatty acid composition of Brevibacterium ammoniagenes was affected by the temperature of growth. As the growth temperature was lowered, the proportion of unsaturated fatty acids increased. The fatty acid synthetase obtained from B. ammoniagense produced oleic acid as well as saturated fatty acids. The ratio of unsaturated to saturated fatty acids synthesized by this enzyme in vitro was dependent on the temperature of the enzyme reaction but not on the growth temperature of B. ammoniagenes from which the enzyme was prepared. These results suggest that the changes of composition in cellular fatty acids reflect the temperature dependence of the fatty acid synthetase.     

Effect of diazepam on the fatty acid composition of plasma and liver phospholipids in rat.

Male Wistar rats (2 months old) were maintained on a nutritionally adequate diet, and diazepam was administered at a dose of 10 mg/kg/day. After 24 weeks the effects on the fatty acid composition of plasma and liver phospholipids were studied. Increased levels of palmitic (16:0), palmitoleic (16:1n-7), stearic (18:0), and oleic (18:1n-9) acids were found in plasma phospholipids. In contrast, the levels of docosapentanoic (22:5n-3) and docosahexanoic (22:6n-3; DHA) acids were drastically decreased by diazepam. A significant decrease produced by diazepam was also found in levels of DHA in liver phospholipids.     

Discrimination of microbial diversity by fatty acid profiles of phospholipids and lipopolysaccharides in differently cultivated soils

The effects of long-term management practices on the diversity of the microbial community were examined by analyzing the composition of fatty acids (FAs) in phospholipids (PL) and lipopolysaccharides (LPS). According to the Principal Component Analysis (PCA) of total fatty acids the soils were divided in two groups: a) Black fallow soil (1) and soils cropped with potatoes (3, 4), and b) green fallow soil (2), soils cropped with wheat (5, 6), crop rotation (7) and grassland (8). The PCA for saturated FAs and for hydroxy FAs of both PL and LPS shows that the green fallow soil (2) can be distinguished from the other soils. For monounsaturated FAs the grassland soil (8) and for polyunsaturated FAs the wheat with vetch soil (6) clearly differed from the other soils. Fatty acids with biomarker quality such as 15:0 for bacteria and 18:26 for fungi were used for determining the ratio between bacteria and fungi: the black fallow soil (1) and the soil managed with crop rotation (7) contained significantly higher proportions of bacteria than the other soils. The largest proportion of the indicator fatty acid il5:0 for Gram-positive bacteria was measured in the black fallow soil (1), while the-hydroxy FAs indicative of Gram-negative bacteria most frequently occurred in manured potato cropped soil (4). Both indicator fatty acids 18:26 for fungi and cy19:0 for anaerobic bacteria had their highest concentrations in the manured potato cropped soil (4).     

Structures of the O-specific polysaccharides and a serological cross-reactivity of the lipopolysaccharides of Proteus mirabilis O24 and O29.

Strains of Proteus mirabilis belonging to serogroups O24 and O29 are frequent in clinical specimens. Anti-P. mirabilis O24 serum cross-reacted with the lipopolysaccharide (LPS) of P. mirabilis O29 and vice versa. The structures of the O-specific polysaccharides (OPSs, O-antigens) of both LPSs were established using sugar analysis and one- and two-dimensional 1H- and 13C-NMR spectroscopy and found to be different. SDS-PAGE and Western immunoblotting suggested that the serological cross-reactivity of the LPSs is due to a common epitope(s) on the core-lipid A moiety, rather than on the OPS. Therefore, the epitope specificity and the structures of the O-antigens studied are unique among Proteus serogroups.     

Transposon mutagenesis in Proteus mirabilis.

A technique of transposon mutagenesis involving the use of Tn5 on a suicide plasmid was developed for Proteus mirabilis. Analysis of the resulting exconjugants indicated that Tn5 transposed in P. mirabilis at a frequency of ca. 4.5 x 10(-6) per recipient cell. The resulting mutants were stable and retained the transposon-encoded antibiotic resistance when incubated for several generations under nonselective conditions. The frequency of auxotrophic mutants in the population, as well as DNA-DNA hybridizaiton to transposon sequences, confirmed that the insertion of the transposon was random and the Proteus chromosome did not contain significant insertional hot spots of transposition. Approximately 35% of the mutants analyzed possessed plasmid-acquired ampicillin resistance, although no extrachromosomal plasmid DNA was found. In these mutants, insertion of the Tn5 element and a part or all of the plasmid had occurred. Application of this technique to the study of swarmer cell differentiation in P. mirabilis is discussed.     

Effects of Proteus mirabilis lipopolysaccharides with different O-polysaccharide structures on the plasma membrane of human erythrocytes

The effects of O33 and O49 P. mirabilis lipopolysaccharides (LPSs) on human erythrocyte membrane properties were examined. Physical parameters of the plasma membrane, such as membrane lipid fluidity, physical state of membrane proteins, and osmotic fragility, were determined. The fluidity of the lipids was estimated using three spin-labeled stearic acids of doxyl derivatives: 5-doxylstearic acid, 12-doxylstearic acid, and 16-doxylstearic acid. All the applied labels locate to different depths of the lipid layer and provide information on the ordering of phospholipid fatty acyl chain mobility. LPSs O49 increased the membrane lipid fluidity in the polar region of the lipid bilayer as indicated by spin-labeled 5-doxylstearic acid. An increase in fluidity was also observed in the deeper region using 12-doxylstearic acid only for O33 LPSs. The highest concentration of O33 LPSs (1 mg/ml) increased the motion of membrane proteins detected by the spin-label residue of iodoacetamide. These results showed different actions of O33 and O49 LPSs on the plasma membrane due to the different chemical structures of O-polysaccharides. P. mirabilis O33 and O49 LPSs did not induce changes in the membrane cytoskeleton, osmotic fragility and lipid peroxidation of erythrocytes. On the other hand a rise in the content of carbonyl compounds was observed for the highest concentrations of O33 LPS. This result indicated protein oxidation in the erythrocyte membrane. Lipid A, the hydrophobic part of LPS, did not change the membrane lipid fluidity and osmotic fragility of erythrocytes. Smooth and rough forms of P. mirabilis LPSs were tested for their abilities for complement-mediated immunohemolysis of erythrocytes. Only one out of seven LPSs used was a potent agent of complement-mediated hemolysis. It was rough, Ra-type of P. mirabilis R110 LPS. The O-polysaccharide-dependent scheme of reaction is presented.     

Fumarate reduction in Proteus mirabilis.

1. Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorate-resistant mutant strains of the wild type, fumarate reductase appeared to be affected. 2. Cytoplasmic membrane suspensions isolated from anaerobically grown P. mirabilis oxidized formate and NADH with oxygen and with fumarate, too. 3. Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochrome b, cytochrome a1 and cytochrome d. Cytochrome b was reduced by NADH as well as by formate to approximately 80%. 4. 2-n-Heptyl-4-hydroxyquinilone-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state. 5. The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochrome b. 6. The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen. 7. Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochrome b as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grown P. mirabilis is presented.     

Genetic regulation of unsaturated fatty acid composition in C. elegans

Delta-9 desaturases, also known as stearoyl-CoA desaturases, are lipogenic enzymes responsible for the generation of vital components of membranes and energy storage molecules. We have identified a novel nuclear hormone receptor, NHR-80, that regulates delta-9 desaturase gene expression in Caenorhabditis elegans. Here we describe fatty acid compositions, lifespans, and gene expression studies of strains carrying mutations in nhr-80 and in the three genes encoding delta-9 desaturases, fat-5, fat-6, and fat-7. The delta-9 desaturase single mutants display only subtle changes in fatty acid composition and no other visible phenotypes, yet the fat-5;fat-6;fat-7 triple mutant is lethal, revealing that endogenous production of monounsaturated fatty acids is essential for survival. In the absence of FAT-6 or FAT-7, the expression of the remaining desaturases increases, and this ability to compensate depends on NHR-80. We conclude that, like mammals, C. elegans requires adequate synthesis of unsaturated fatty acids and maintains complex regulation of the delta-9 desaturases to achieve optimal fatty acid composition.     

Structure, function, and regulation of Escherichia coli rRNA in Proteus mirabilis.

Escherichia coli rRNA genes have been introduced into Proteus mirabilis on an F-prime factor (F'14). A portion of the ribosomes in the resulting merodiploid consist of E. coli rRNA and P. mirabilis ribosomal proteins. These ribosomes are structurally similar to normal P. mirabilis or E. coli ribosomes and exhibit many or all of the functional properties of normal ribosomes. The accumulation of E. coli rRNA in the merodiploid is regulated in a way similar to the the regulation of P. mirabilis rRNA.     

Fatty acid composition of the milk lipids of Nepalese women: correlation between fatty acid composition of serum phospholipids and melting point.

Milk was collected from 36 Nepalese women, 15 to 32 years of age, in order to investigate relationships between the proportions of intermediate chain-length (C10-C14) fatty acids and critical n-3 and n-6 polyunsaturated fatty acids in the milk lipids they were producing. Serum was also obtained from these lactating women and the fatty acid composition of their serum phospholipid fraction was determined and compared with that of the corresponding milk lipid fraction. Compared to women in technologically advanced parts of the world, the serum phospholipids of the Nepalese women contained nutritionally adequate proportions of linoleic acid (LA) (16.8%), alpha-linolenic acid (ALA) (0.53%), arachidonic acid (AA) (5.69%), and docosahexaenoic acid (DHA) (1.42%). However, although the milk lipids contained adequate proportions of ALA (1.81%), AA (0.43%), and DHA (0.23%), the lipids contained low to moderate percentages of LA (mean, 9.05%). Positive correlations were observed between the proportions of AA (P=0.001, r=0.50) and ALA (P=0.03, r=0.36) in the serum phospholipids and milk lipids of the women. As the proportion of C10-Cl4 fatty acids in the milk lipids increased from 10% to 40%, there was preferential retention of three critical n-3 and n-6 fatty acids (ALA, AA, and DHA) at the expense of two relatively abundant nonessential fatty acids, namely stearic acid and oleic acid. In addition, using fatty acid melting point data and the mol fraction of the 9 most abundant fatty acids in the milk, we estimated the mean melting point (MMP) of the milk lipids of the Nepalese women. The MMPs ranged from 29.3 to 40.5 degrees C (median, 35.5 degrees C). These results indicate that: 1) the levels of AA and ALA in the blood of lactating mothers influence the levels of these fatty acids in the milk they produce; 2) when the mammary gland produces a milk that is rich in C10-Cl4 fatty acids, it somehow regulates triglyceride synthesis in such a way as to ensure that the milk will provide the exclusively breast-fed infant with the amounts of the critical n-3 and n-6 fatty acids it requires for normal growth and development; and 3) the melting point of the milk lipid fraction is determined mainly by the mol % of the intermediate chain-length (C10-C14) fatty acids, oleic acid, linoleic acid, and alpha-linolenic acid.