One-step purification of bacterial lipid macroamphiphiles by hydrophobic interaction chromatography.

Bacterial lipid macroamphiphiles extracted with phenol/water can be purified in one step by hydrophobic interaction chromatography. Lipids and the major part of protein are separated from macroamphiphiles during phenol/water extraction. Coextracted nucleic acids, polysaccharides, and residual protein are effectively removed by column chromatography on octyl-Sepharose whereby macroamphiphiles are primarily adsorbed and later eluted with a buffered propanol gradient. The procedure is applicable to macroamphiphiles with various lipid structures as was demonstrated using the diacylglycerol-containing lipoglycan of Micrococcus luteus, the lipid A-containing lipopolysaccharide of Salmonella typhimurium, and the diglyceryl tetraether lipoglycans of Thermoplasma acidophilum and Thermoplasma volcanicum. On elution from octyl-Sepharose, separation into molecular species of different compositions was observed with the lipopolysaccharide of S. typhimurium and the lipoglycan of T. volcanicum. It was also shown that, after phenol/water extraction, membrane lipids are completely recoverable from the phenol layer, which makes it possible to isolate lipids along with macroamphiphiles from the same sample of bacteria.

The amino terminus of the aspartate chemoreceptor is formylmethionine.

The amino terminus of the Salmonella typhimurium aspartate receptor has been identified as formylmethionine by mass spectral analysis of the amino-terminal tryptic peptide. Purification and analysis of the blocked amino-terminal peptide was facilitated by the use of a mutant aspartate receptor which has a cysteine residue at position 3. The sequence of this peptide confirms the translational start site predicted from the nucleotide sequence of the tar gene. Furthermore, in vivo labeling experiments reveal that the formyl group is present on chemotaxis receptors produced at wild-type levels in Escherichia coli, indicating that the presence of the formyl group is not a consequence of over-production of the receptor. The stability of the amino-terminal formyl group on the receptor may be a consequence of the membrane localization of the receptor and the dependence of this localization on the membrane transport machinery of the cell.

Interactions between Salmonella typhimurium lipopolysaccharide and the antimicrobial peptide, magainin 2 amide.

Effects of magainin 2 amide on the phase behavior of Salmonella typhimurium lipopolysaccharide were characterized by FT-IR spectroscopy. This antimicrobial cationic peptide disorders the lipopolysaccharide at molecular ratios of lipopolysaccharide to magainin greater than 4, and can induce a temperature-dependent structural reorientation. The nature of the five phosphate groups of lipopolysaccharide was determined by 31P NMR spectroscopy. At pH 7.4, the net charge on the phosphates is -7. Lipopolysaccharide undoubtedly plays an important role in modulating the interactions of magainin with the gram-negative cell envelope and may act as a molecular sponge to protect the plasma membrane.

Purification of the cysB protein from Salmonella typhimurium.

Using an assay based on polypeptide mobility in one- and two-dimensional polyacrylamide electropherograms, we purified the Salmonella typhimurium cysB protein from an Escherichia coli strain carrying plasmid pGBK14, in which the S. typhimurium cysB gene is under transcriptional control of the strong promoter PL from phage lambda. cysB protein constitutes approximately 4% of total soluble protein in such cells and was obtained with good yield at a purity of 85% after precipitation of nucleic acids with streptomycin sulfate, ammonium sulfate fractionation, and hydrophobic chromatography on methyl agarose. Material with 95% purity was obtained with poor yield by ion-exchange chromatography of native protein and with good yield by size exclusion chromatography of denatured protein in sodium dodecyl sulfate. Physicochemical studies of the purified cysB protein show that it is a tetramer of identical 36-kDa subunits with a pI, amino acid composition, amino-terminal sequence, and carboxyl-terminal amino acid content identical to those known from previous studies or deduced from the cysB DNA sequence. Although O-acetyl-L-serine is believed to bind to cysB protein to form a complex that stimulates transcription of genes of the cysteine regulon, we were unable to demonstrate such an interaction using methods that would have detected binding with a Kd of less than 0.1 mM.

Luciferin-luciferase assay of adenosine triphosphate from bacteria: a comparison of dimethylsulphoxide (DMSO) and acetone with other solvents.

The extraction of adenosine triphosphate (ATP) from six strains of bacteria, chosen for differences in cell-wall composition and habitat, was performed. The solvents were two in common use, Tris-ethylenediaminetetraacetate (Tris-EDTA) and trichloroacetic acid (TCA), and two promising, though less utilized solvents, dimethylsulphoxide (DMSO) and acetone. ATP was determined by the luciferin-luciferase reaction. Of the solvents used, DMSO and acetone were the most effective considering the different kinds of bacteria tested and, of these two, DMSO was the most convenient to use. Tris-EDTA was not as effective as the other solvents tested and TCA, which was effective with most strains, gave low yields when used with cultures grown in artificial seawater broth. Internal standards were used to determine if there were substances present that could inhibit the reaction of released ATP with the luciferin-luciferase reagent. Extracts of ATP, stored at -20 degrees C, were stable for up to 3 weeks.

Global control in Salmonella typhimurium: two-dimensional electrophoretic analysis of starvation-, anaerobiosis-, and heat shock-inducible proteins.

The response of Salmonella typhimurium to various forms of environmental stress was examined by using O'Farrell two-dimensional gel electrophoresis. Polypeptides (a total of 110) which quantitatively increased during various starvations, anaerobiosis, or heat shock were identified and cataloged in reference to a standard polypeptide map. Although significant overlap was noted during comparison of proteins induced by different starvations, only a few proteins produced during heat shock or anaerobiosis were also identified as starvation inducible.

Gas chromatographic determination of the fatty acid composition of endotoxins from different bacteria.

Endotoxins from four bacterial species extracted by three different procedures were acid-methanolyzed and the methyl esters of the fatty acids were analyzed by packed-column gas chromatography. There were qualitative and quantitative differences in the fatty acid profiles of the lipopolysaccharides isolated from four Gram-negative bacteria. Our data show considerable lot-to-lot variations in amounts of four methyl esters from the same bacterial serotype extracted by the same procedure and in the same bacterial serotype extracted by different procedures. These results indicate that extraction and perhaps culture conditions, as well as bacterial species, affect the fatty acid composition of endotoxins, hydrolyzed and derivatized by these procedures.

[Bioassay for the detection of hydroxamate-iron-chelators (Aerobactin)]. / Biotest zum Nachweis von Hydroxamat-Fe-Chelatoren (Aerobactin).

Different Salmonella strains were tested for aerobactin production in a hydroxamate-bioassay with the aerobactin indicator strain E. coli LG 1522. The majority of hospital strains of Salmonella typhimurium produce hydroxamate siderophore. On the other hand S. typhimurium strains belonging to phage type n. c. 1/72/n. c., biochemical type b from human and animal sources, were unable to produce this siderophore. Serotypes other than S. typhimurium for example the multiresistent S. wien hospital strains, which were isolated in western europe and in the GDR, can excreate hydroxamate siderophore. Plasmids pIE 528 and pIE 5 234 isolated from Salmonella hospital strains produce hydroxamate siderophore in the enterobactin negative Salmonella typhimurium-strain enb-7. Thus, the hydroxamate bioassay may be a useful supplementary test for epidemiological strain characterization.

A diffusion assay for detection and quantitation of methyl-esterified proteins on polyacrylamide gels.

The methyl esterification of bacterial and mammalian proteins is a subject of increasing interest and effort. Such studies in intact cells typically involve the use of [methyl-3H]methionine which is taken up and incorporated into S-adenosyl-L-methionine, the methyl donor. The level of methylation, however, is much less than the incorporation of labeled methionine directly into protein. A diffusion assay which distinguishes [3H]methionine from the base-labile [3H]methyl esters is described here. The ester linkage is hydrolyzed at high pH to release [3H]methanol from the sample which diffuses into an adjacent pool of scintillation fluid. The assay is contained in a scintillation vial which can be counted directly.

Stimulation of spleen cells and macrophages of C3H/HeJ mice by a lipid A precursor derived from Salmonella typhimurium.

Lipid A is that portion of the lipopolysaccharide (LPS) molecule believed to mediate most of the biologic activities associated with protein-free endotoxic preparations. C3H/HeJ mice possess a mutation at the Lps gene locus (Lpsd) that results in a state of profound hyporesponsiveness to the biologic effects of LPS (and more specifically, lipid A) in vivo. The relative unresponsiveness in vivo to LPS exhibited by these mice is reflected at a cellular level, as evidenced by a failure of many different cell types derived from the C3H/HeJ strain to respond to LPS or lipid A in vitro; this lack of response contrasts with that of cell cultures prepared from endotoxin responsive (Lpsn) mouse strains. Evidence is presented which demonstrates that a lipid A precursor molecule, produced by a mutant of Salmonella typhimurium conditionally defective in the synthesis of 3-deoxy-D-mannooctulosonic acid, stimulates mitogenesis in C3H/HeJ splenic cultures and induces cultures of C3H/HeJ macrophages to produce significant levels of the monokine interleukin-1 (IL-1; previously referred to as lymphocyte activating factor or LAF) and prostaglandins of the E series. These findings suggest the possibility that the failure of C3H/HeJ cells to respond to intact LPS or lipid A may be related to a defect in the processing of lipid A or LPS to a suitably stimulatory form, rather than to a defect in the recognition of the lipid A region.

Lipid A and immunotherapy.

Endotoxin isolated from Re mutants of Salmonella typhimurium or Salmonella minnesota and consisting only of 3-deoxy-D-mannooctulosonic acid (KDO) and lipid A synergistically enhances the ability of mycobacterial cell wall skeleton (CWS) to regress transplantable, line-10 tumor (hepatocellular carcinoma) in syngeneic guinea pigs. Tumor regression is rapid, and systemic tumor immunity concomitantly develops when as little as 50 micrograms of each of these two components is combined and injected intralesionally. Selective removal of KDO from endotoxin yields diphosphoryl lipid A, which retains its toxic properties. Subsequent selective removal of the phosphate moiety at the reducing end of the diphosphoryl lipid A molecule yields nontoxic, monophosphoryl lipid A (determined by lethality for chick embryos). Like the parent endotoxin or toxic diphosphoryl lipid A, monophosphoryl lipid A retains the ability to synergistically enhance the antitumor activity of mycobacterial CWS adjuvant. Both di- and monophosphoryl lipid A contain mixtures of a series of structural analogs. They can be separated chromatographically into single components that differ in number, type, and position of ester-linked fatty acids. Comparison of chromatographic fractions reveals that components of toxic and nontoxic lipid A can be paired according to structure. Each component of the pair has the same molecular structure, with the exception of an additional phosphate group in the toxic component. The toxicity of "lipid A's" liberated from endotoxin by acid hydrolysis appears to be determined by the proportion of di- and monophosphoryl lipid A in the hydrolysis mixture. Structural analogs of monophosphoryl lipid A, which differ in degree of O-acylation and type and distribution of fatty acids, have comparable antitumor activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of macrophage-mediated tumor cell killing by bacterial outer membrane proteins (porins).

Various microbial products are known to influence the function of mouse peritoneal macrophages. Lipopolysaccharide (LPS) and certain lipid A-associated proteins are known to enhance the tumoricidal effects of macrophages. The purpose of this study was to determine whether porins (outer membrane proteins) of Salmonella typhimurium G30/C21 would influence the activity of macrophages from lipid A-responsive and -unresponsive mice. Porins, extracted by a combined sodium dodecyl sulfate-EDTA method from cell walls, were free of LPS as determined by Limulus amebocyte lysate assay and appeared as a band at approximately 36,000 molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In tumor cell killing assays done under LPS-free conditions, the porins in doses of 1 to 10 ng/ml enhanced the tumoricidal effect of macrophages from bacillus Calmette-Guérin-infected C3H/HeN or C3H/HeJ mice. Protein-free LPS enhanced the tumoricidal activity of macrophages from bacillus Calmette-Guérin-infected C3H/HeN but not C3H/HeJ mice. The tumoricidal-enhancing activity of protein-free LPS was blocked by the lipid A-binding antibiotic polymyxin B sulfate, but the effects of porins were not altered by the polymyxin B sulfate. These results suggest that porins, proteins known to alter membrane function, may alter macrophage function by interaction with macrophage membranes.

Diadenosine 5',5"'-P1,P4-tetraphosphate and related adenylylated nucleotides in Salmonella typhimurium.

Salmonella typhimurium LT2 rapidly accumulates high levels of a family of five adenylylated nucleotides following exposure to a bacteriostatic quinone, 6-amino-7-chloro-5,8-dioxoquinoline. These compounds have been analyzed using our recently described two-dimensional thin layer chromatographic method. The five dinucleotides, which cannot be detected in exponentially growing cells, have been identified as diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA), ApppGpp (guanosine 3'-diphosphate-5'-adenosine-5'-(P1,P3-triphosphate)), AppppG (adenosine 5'-guanosine-5'-(P1,P4-tetraphosphate)), ApppG (adenosine 5'-guanosine-5'-(P1,P3-triphosphate)), and ApppA (diadenosine 5',5"'-P1,P3-triphosphate). AppppA has been previously detected in vitro as an enzymatic product of aminoacyl-tRNA synthetases and in vivo at submicromolar levels in eucaryotic cells. The induced intracellular concentration of AppppA and the other adenylylated nucleotides in S. typhimurium is approximately 100-fold higher than that found in eucaryotic cells. We propose that these dinucleotides are alarmones, regulatory molecules signaling a particular metabolic stress.

Structural requirements of endotoxic glycolipid for antitumor and toxic activity.

Endotoxic glycolipids extracted from the polysaccharide heptose-less Re mutant of Salmonella typhimurium were hydrolyzed with alkaline and acid reagents. Treatment with hydroxylamine caused the liberation of all O-ester linked fatty acids and resulted in abrogation of the toxicity (lethality to chick embryos) and ability to regress tumors (line-10 tumors in strain 2 guinea pigs). Treatment with dilute sodium hydroxide caused partial removal of O-ester linked fatty acids without loss of these activities. Toxicity and tumor-regressive potency were retained after removal of 2-keto-3-deoxyoctonate (KDO) by exposing the glycolipids to sodium acetate solution at pH 4.5. The majority of the glycolipids of the endotoxic extracts were rendered non-toxic but retained antitumor activity when hydrolyzed with boiling 0.1 N hydrochloric acid, which split KDO and glycosidic phosphate from the glycolipid molecules. Non-toxic glycolipid fractions possessing antitumor activity were separated from the acid hydrolysate by means of preparative thin layer chromatography. It was concluded that glycosidic bound phosphate and at least a portion of the fatty acids of the lipid A moiety are essential for toxicity, but that this phosphate is not an essential structural feature for tumor-regression activity.

Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium.

Endotoxin extracted from the heptose-less mutant of Salmonella typhimurium was hydrolyzed in 0.1 N HCl in methanol/water (1:1, v/v) at 100 degrees C to yield lipid A, which was then fractionated on a Sephadex LH-20 column to yield a major monophosphoryl lipid A fraction. The monophosphoryl lipid A was further fractionated by preparative thin layer chromatography. This process yielded three major bands (TLC-1, -3, and -5) and two minor bands (TLC-7 and -9). The purity of these fractions was established by ion exchange and reverse phase high performance liquid chromatography. The thin layer fractions were analyzed by fast atom bombardment mass spectrometry. TLC-1 and -3 gave molecular ions (M-H)- at m/e 1730 and 1716, respectively. Both of these fractions contained beta-hydroxymyristic, lauric, and 3-myristoxymyristic acids in O-acyl linkages. The molecular formula and Mr of TLC-1 are C95H179O22N2P and 1731.16; those of TLC-3 are C94H177O22N2P and 1717.15. TLC-1 was a methyl homolog of TLC-3. The major component of TLC-5 (C80H151O22N2P and Mr = 1506.99) gave a molecular ion at m/e 1506 and contained two beta-hydroxymyristic acids and a lauric acid in the O-acyl linkages. The major component of TLC-7 (C66H125O19N2P and Mr = 1280.83) and the single component of TLC-9 gave molecular ions at m/e 1280 and 1098, respectively. TLC-7 contained lauric and beta-hydroxymyristic acids in the O-acyl linkages. TLC-9 (C54H103O18N2P and Mr = 1098.69) contained a single O-acylated beta-hydroxymyristate group. TLC-1 and -3 were nontoxic in the chick embryo lethality test and regressed established tumors in the syngeneic guinea pigs.

cis 2-Methylthio-ribosylzeatin (ms2io6A) is present in the transfer RNA of Salmonella typhimurium, but not Escherichia coli.

We have identified the cis isomer of N6-(4-hydroxy-isopentenyl)-2-methylthioadenosine (ms2io6A) as a component of the tRNA of Salmonella typhimurium. This is the first report of this compound in the tRNA of any member of the enterobacteriaceae: the nucleoside was previously thought to be found exclusively in plants or plant associated bacteria. Interestingly, all E. coli strains examined were found to lack ms2io6A. Evidence is presented which suggests S. typhimurium tRNA also contains low levels of 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U) in addition to 5-methylaminomethyl-2-thiouridine (mnm5s2U).

The transfer RNA of certain Enterobacteriacae contain 2-methylthiozeatin riboside (ms2io6A) an isopentenyl adenosine derivative.

Isopentenyl adenosine derivatives are always located adjacent to the 3' end of the anticodon in transfer RNA and have been implicated in certain biological functions. In the enteric bacterium, E. coli, the derivative is ms2i6A whereas in some plant associated bacteria the derivative is the hydroxylated form, ms2io6A. Anti-i6A immunoadsorbent chromatography has been employed to detect isopentenyl adenosine compounds. In the present study we show that the transfer RNA of three species of enteric bacteria, S. typhimurium, K. pneumoniae, and S. marcescens contains both ms2io6A and ms2i6A. Under the growth conditions utilized the ms2io6A is predominant. The presence of ms2io6A in Enterobacteriacae is particularly noteworthy since in previous work it has been found only in plant-associated species of bacteria.

Osmotic shock fluids from salmonella R-mutants. Chemical composition and protective capacity against S. typhimurium infection in mice.

Salmonella R-mutants of different chemotype were subjected to osmotic shock treatment according to the method of Willis et al. (18). Chemical composition (protein, LPS, palmitic acid and nucleic acids) as well as the polypeptide patterns of the shock fluid and hypertonic fluid (in which the cells were suspended before shock treatment) were determined and compared with material extracted by urea treatment carried out to get protein from the cell surface. The results indicate that even components from the cell surface have been released by shock treatment. All the extracts mentioned above showed protective activity in mouse against infection.

Essential regions of the lipopolysaccharide of Pseudomonas aeruginosa responsible for pyrogenicity and activation of the proclotting enzyme of horseshoe crabs. Comparison with antitumor, interferon-inducing and adjuvant activities.

Regions of lipopolysaccharide derived from Pseudomonas aeruginosa essential for pyrogenicity and activation of the proclotting enzyme of the horseshoe crab were examined. Free lipid A with intact fatty acids showed strong pyrogenicity but showed little activation of the proclotting enzyme. Chemical modification of the polysaccharide portion and deacylation of the lipopolysaccharide diminished activation of the proclotting enzyme. The native-protein portion attached to the lipopolysaccharide also inhibited the activation of proclotting enzyme by lipopolysaccharide, but not pyrogenicity. These results indicate that free lipid A is sufficient for pyrogenicity, whereas the complete lipopolysaccharide is the strongest activator of the proclotting enzyme. The lipopolysaccharide of P. aeruginosa, which showed the strongest activation of proclotting enzyme, showed the weakest pyrogenicity of all the lipopolysaccharides tested here. All these results demonstrate that there is not correlation between pyrogenicity and proclotting enzyme activation induced by lipopolysaccharides.

Protective activity of extracts from Salmonella-R-mutants against Salmonella typhimurium infection in mice. Influence of repeated extractions on the chemical composition and efficacy of the extracts.

Repeated extractions (three times) of an S-form and different R-mutants of S. typhimurium and S. minnesota with urea medium resulted in extracts varying in their chemical composition with respect to protein, lipopolysaccharides, phospholipids and nucleic acids. Protein appeared to be the major component of all the extracts and was present in substantial amounts in the third extract also. With subsequent extractions the lipopolysaccharide and phospholipid contents generally decreased in the R-mutants, but increased in the S-form. The nucleic acid contents always increased with repeated extractions. polypeptide patterns of the materials obtained after repeated extraction showed some differences in case of R-mutants predominantly. Active immunization of mice with these extracts protected them almost to the same extent against S. typhimurium infection. Only in case of one R-mutant a graded decrease in protection could be observed from extract 1 to extract 3. However, the differences in composition of the vaccines based on chemical analysis data and polypeptide pattern could not account for this decrease in protection.