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A new CE method for fast and efficient analysis of bacterial endotoxins (lipopolysaccharides) is described. It is based on the strong interaction between proteins and endotoxins. The UV absorption of the protein component in the complex is used for the detection. The electrophoretic mobility of the complex hemoglobin/endotoxin can be employed for qualitative analysis of the endotoxin. For instance, the structural differences between "smooth" and "rough" lipopolysaccharides from Salmonella minnesota (wild-type), Salmonella minnesota R595 and Shigella sonnei R562H are reflected in the electrophoretic mobilities of their hemoglobin complex.

To investigate the role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis synovial specimens from 92 patients were analysed for the presence of bacterial DNA with the use of polymerase chain reaction and for the presence of lipopolysaccharide and enterobacterial common antigen (ECA) with the use of Dot-ELISA. In addition, peripheral blood samples were available for PCR analysis from 68 patients. Salmonella and Yersinia chromosomal DNA was not found in any of the synovial specimens and blood samples from the patients. All of the synovial fluids were also culture-negative. Salmonella LPS antigens were observed in 8 (8.6%), Yersinia in 20 (21.7%) and ECA antigens in 32 (34.9%) synovial specimens. Our findings revealed the presence of bacterial degradation products, but not bacteria from the genus Salmonella and Yersinia or their DNA in the synovial fluid or blood of patients with spondyloarthropathies and rheumatoid arthritis.

Aims: To relate the diseases caused by strains of Salmonella paratyphi B and S. java to pathogenic mechanisms expressed by these bacteria for the purpose of organism discrimination., Methods and Results: Epidemiological data relating to cases of disease caused by strains of S. paratyphi B and S. java, isolated over a 10-year period, were analysed with respect to patients' symptoms, particularly those involving enteric fever. Strains of S. paratyphi B and S. java were also examined for a range of known pathogenic mechanisms. Infection with S. paratyphi B involved pyrexia in 12.5% of patients compared with 2.2% of patients infected with S. java. These organisms could not be differentiated based on the pathogenic properties examined., Conclusions: Strains of S. paratyphi B appear not to be a major cause of enteric fever but primarily a cause of gastroenteritis, in common with S. java. Both organisms express similar pathogenic mechanisms, and strains of S. java are probably d-tartrate utilizing variants of S. paratyphi B., Significance and Impact of the Study: Strains of S. paratyphi B are very closely related organisms, primarily causing gastroenteritis. From this study it would appear that strains of S. paratyphi B are not a major cause of enteric fever.

Aims: We have recently reported the isolation of Salmonella that have acquired tolerance to hypochlorous acid (HOCl) (Mokgatla et al. 1998). The aim of this work was to investigate possible protective mechanisms involved in the increased tolerance to HOCl of a selected resistant strain., Methods and Results: One resistant (Salmonella 104) and one sensitive (Salmonella 81) isolate in exponential phase were exposed to HOCl at a final active concentration of 28 mg l(-1). Cultures were assayed for superoxide dismutase and catalase activity, as well as for four membrane-bound dehydrogenases (malate, lactate, glutamate and glucose-6-phosphate dehydrogenase). The degree of single-strand breaks in genomic DNA was analysed and lipopolysaccharide profiles determined. The resistant Salmonella isolate differed from the sensitive isolate in a number of ways. It responded within 10 min of exposure by producing catalase and decreasing the activity levels of four membrane-bound dehydrogenases. This combination would lead to lower levels of hydroxyl radicals and singlet oxygen, moieties thought to be integrally involved in the antibacterial action of HOCl. Furthermore, the resistant strain did not display the same degree of DNA damage as did the sensitive strain., Conclusions: Strain 104 is believed to grow in the presence of 28 mg l(-1) HOCl by protecting itself against HOCl by decreasing the levels of species that could react with HOCl to generate toxic reactive oxygen radicals and by improved DNA damage repair mechanisms., Significance and Impact of the Study: The occurrence of Salmonella able to grow in the presence of 28 mg l(-1) HOCl is of relevance to the food-processing and drinking water treatment industries as these strains would survive sanitation regimes.

TNF-alpha mRNA induction in murine macrophages by virulent and avirulent Salmonella strains was measured in vitro by RT-PCR method. Virulence plasmid-cured strains of S. choleraesuis serovar Typhimurium and serovar Choleraesuis, and rpoS-defective mutant of S. choleraesuis serovar Typhimurium induced significantly higher level of TNF-alpha mRNA than their parent (virulent) strains in macrophages of C3H/HeN mice. When macrophages of LPS-low responder (C3H/HeJ) mice were used, the difference of the induction level was not observed, indicating that LPS was involved in the enhanced level of TNF-alpha mRNA induction by avirulent Salmonella strains. LPSs from virulent and avirulent strains were analysed, but no difference was found for cytokine-inducing activity, and chemical properties. Those results suggested that avirulent Salmonella strains were damaged more easily, and released more LPS in macrophages to enhance TNF-alpha induction.

Strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow, carrying different numbers of plasmids, were examined for the ability to multiply in sera. Viable counts were performed to monitor the kinetics of growth of bacteria when in human, chicken and turkey sera. The presence of plasmids in Salm. enteritidis, Salm. typhimurium and Salm. virchow reduced considerably the ability of strains of these serotypes to multiply in serum. SDS-PAGE was used to show that growth of Salm. enteritidis in serum did not involve changes in outer membrane proteins or lipopolysaccharide. It was concluded that the carriage of plasmids may be disadvantageous for the survival in serum of certain common salmonella serotypes.

Two indirect ELISA containing outer membrane protein (OMP) and lipopolysaccharide (LPS) antigens from a field isolate of Salmonella choleraesuis var kunzendorf were developed and evaluated in experimentally infected and uninfected control pigs. Experimentally induced infection with S choleraesuis was successfully established in 10 pigs by oral inoculation with 10(8) organisms, and 3 pigs died of clinical salmonellosis at postinoculation (PI) weeks 1, 2, and 4. Swab specimens from tonsils, nostrils, and rectum of pigs were obtained for culture, and sera were evaluated at weekly intervals for 9 weeks after inoculation. The ELISA containing OMP and LPS antigens with either anti-swine IgG or protein albumin-to-globulin ratio (antiglobulin) conjugates were standardized for serologic evaluation. All 4 ELISA (2 OMP and 2 LPS) detected seroconversion by PI week 3 and had sensitivities and specificities of 97.8 and 88.8, 100 and 100, 95.6 and 88.8, and 93.3 and 72.5%, at their ideal cutoff points (negative mean optical density +2 SD). There was excellent agreement between all 4 ELISA systems as determined by kappa values. Cultures of fecal, tonsil, and nasal swab specimens were positive for S choleraesuis until the fourth week of infection. Fecal swab specimens from 1 pig were positive for S choleraesuis until PI week 7. Persistent infection after antemortem culture results were negative was detected by all 4 ELISA, which indicated consistently high titers until the end of PI week 9.(ABSTRACT TRUNCATED AT 250 WORDS)

Ten Salmonella strains belonging to five serological groups were cultivated both at 37 degrees C and close to their individual temperature minima at 12-14 degrees C and the composition of their cell wall lipopolysaccharides (LPS) was compared. When grown at low temperature, the proportion of unsubstituted (free) R-LPS in the total LPS moiety increased significantly in 6 strains, whereas in the other strains, no change or even a slight decrease in the R-LPS proportion was observed as judged from the analyses by SDS-PAGE. In the immunoblot, the R-LPSs from 9 out of 10 strains showed a modified reactivity against a set of specific Salmonella R antisera (anti-Ra, anti-Rb1, anti-Rb2, anti-Rc). In most cases, the decrease in Rb1 reactivity was paralleled by an increase in Rb2 reactivity and also by an increase in the total amount of free R-LPS. The electrophoretic mobility of free R-LPS was changed in 7 out of 10 strains, although the changes were not unidirectional. All changes occurred only in the range of the Ra-Rb1 chemotypes and no significant correlation to the serological grouping of the strains was evident. When grown at low temperature, the average number of O-repeating units was reduced in the majority of cases and in some cases, also the banding profile in SDS-PAGE in the S-LPS region was modified. The fatty acid spectra showed some changes which were in accordance with previous results, namely a decrease in the content of C-12:0 and C-16:0 and an increase in that of C-14:0 and C-16:1. The different influences of the growth temperature on the LPS biosynthesis of different Salmonella strains may be a result of the genetic diversity of this group of microorganisms.

Various chemotypes (Re, Rd2, Rd1P-, Rd1, RcP-, Rc, Rb3, Rb2, Rb1, and Ra) of R-form lipopolysaccharides (LPSs) of Salmonella spp. were crystallized by treatment with 70% ethanol containing 250 mM MgCl2, and crystals of the LPSs were observed electron microscopically and analyzed by electron diffraction and synchrotron X-ray diffraction. All the LPSs tested formed three-dimensional crystals showing very similar shapes; hexagonal plate, solid column, discoid, square or rectangular plate, lozenge plate and truncated hexangular or rectangular pyramid forms. Electron diffraction patterns from the hexagonal plate crystals of all these LPSs obtained by electron irradiation from the direction perpendicular to the basal plane showed that they consist of hexagonal lattices with the lattice constant of 4.62 A. The crystals of all the LPSs thus formed gave ring-like X-ray diffraction patterns because of their small sizes. The long-axis values were calculated from the X-ray diffraction patterns from crystals of all the LPSs in the low-angle region and they corresponded roughly to the length of the proposed primary chemical structures of the R cores of the LPSs. The volume occupied by a single molecule of all the LPSs were calculated from the molecular weights based on the proposed structures and the crystallographic data obtained by electron diffraction, X-ray diffraction, and density determination.

Salmonella abortusovis is a pathogenic bacterium highly specific to sheep, causing spontaneous abortion. In order to understand the role of genes involved in pathogenicity, we investigated S. abortusovis with the random mutagenic TnphoA transposon. A total of 95 S. abortusovis TnphoA mutants yielding alkaline phosphatase active fusion protein were obtained. In this way we created a bank of strains in order to identify any phenotypic modification which could affect the periplasmic and/or exported proteins involved in virulence. The TnphoA mutants were screened for the ability to adhere to epithelial cells: a total of 23 mutant strains lost this phenotypic feature. To detect the chromosomal TnphoA insertions, DNA was restricted by the enzyme EcoRV, which does not cleave the TnphoA sequence. Southern blotting analysis revealed the existence of four classes of integration. Colonies of adhesiveless mutants appear to be as smooth as the S. abortusovis wild type, and electrophoretic analysis indicates a normal lipopolysaccharide profile. To identify mutations affecting genes encoding for outer membrane proteins (OMPs), the alkaline phosphatase portion of the fusion proteins was revealed in TnphoA mutants by immunoblotting with specific antibodies. A mutation in OMPs was detected in seven mutants. Restriction analysis identified in four mutants a common region of 2 kb where alterations in genes coding for OMPs occur. We suggested that this region is involved in pathogenicity in mice, since a group of mutant strains has shown reduced virulence in mice and one mutant is completely avirulent. Furthermore, after mice were exposed orally to these mutants, significant protection against oral challenge with the parental virulent strain resulted.

This study determined the ability of eight strains of Salmonella and their agarsubcultured variants to colonize the intestinal tract of broiler chicks. Nalidixic-acid (NAL)-resistant and streptomycin-resistant subcultured strains (S. california 1989/A and S. typhimurium 3366/A) that persisted in the ceca of chicks in lower numbers than their NAL-resistant parent strains (1989/O and 3366/O) were selected for additional study S. typhimurium strain 3366/A was present in the ceca of chicks in lower numbers than the parent strain 3366/O when given concomitantly with the parent strain or when the two strains were given separately to different chicks. S. california 1989/A strain was present in the ceca in lower numbers than the parent strain after concomitant oral or intracloacal inoculation. Strains 3366/O and 3366/A of S. typhimurium differed in growth rates in BHI broth and cecal mucus. The lipopolysaccharide (LPS) profile indicated that LPS components present in S. california 1989/O were missing from strain 1989/A. A mutant of 1989/O--2095/R--was also LPS- and colonization-deficient.

A radiolabeled lipopolysaccharide (LPS) from Salmonella choleraesuis was as toxic for galactosamine-treated mice as the unlabeled preparation. After intravenous injection, a large proportion (37%) of the labeled material remained in the circulation. This circulating form of LPS was not toxic, even when reextracted with phenol, and remained in the circulation when reinjected into mice. Furthermore, toxic and nontoxic constituents were directly isolated in vitro by hydrophobic gel chromatography of the radiolabeled LPS preparation. The nontoxic form, of lower molecular weight according to its electrophoretic migration, did not react with an anti-lipid A monoclonal antibody but reacted with polymyxin B and was as active on pre-B and B cells as the unfractionated LPS preparation. The results of this study suggest that some LPS constituents of phenol-extracted endotoxin preparations are not toxic for galactosamine-sensitized mice and are not trapped by appropriate LPS-binding sites in the liver as efficiently as are the toxic forms.

A murine hybridoma cell line that produces monoclonal antibody (mAb) against the serogroup D1 Salmonella lipopolysaccharide (LPS) antigen was established. The trisaccharide tyvelose alpha 1----3 mannose alpha 1----4 rhamnose was shown to be involved in the reactive epitope of the mAb since this mAb reacted strongly with strains of serogroup D1 Salmonella but not with Salmonella strains from the O serogroups of A, B, and D2, and sodium meta-periodate was found to destroy the reactivity of the serogroup D1 O-antigen with the mAb. As such this mAb was found to be a useful serotyping reagent for the identification of serogroup D1 Salmonella, and for the differentiation of strains of serogroups D1 and D2 Salmonella which have identical flagellar H antigens.

Using transposon insertion mutagenesis with Tn1 or Tn5, we obtained Salmonella dublin mutant strains that showed either diminished serum resistance (five mutants) or diminished mouse lethality (two mutants). Detailed restriction cleavage analysis to determine the single sites of transposon insertion in an 80 kb plasmid (pTE800) indicated that a region for serum resistance was located within a 3.0 kb region of the SalI cleavage fragment 5 and the HindIII fragment 2, while the region for mouse lethality was within a 6.0 kb region of the SalI fragment 2 and the HindIII fragment 1. When the Tn1-containing SalI fragment 5 was reconverted, by homologous recombination, to the original SalI fragment 5 (9.6 kb), serum resistance was recovered to the same level as that of a parent strain 52401. Moreover, the change in the serum resistance correlated with changes in the neutral sugar composition of the LPS. The mutation in the plasmid in strain TE4-55 that gave diminished mouse lethality was also reversed by recombination with the cloned SalI fragment 2 (15.0 kb), with concomitant recovery of mouse lethality. These results indicate that the genetic region for serum resistance is different from that for mouse lethality, and that the gene for serum resistance is closely involved with the expression of the neutral sugar composition of the LPS of S. dublin.

The phase structure of isolated bacterial lipid A, the lipid anchor of the lipopolysaccharides of the outer membrane of Gram-negative bacteria, has been investigated by neutron small-angle scattering. The shape of the scattering curves obtained at different H2O/2H2O ratios revealed a lamellar organisation of the lipid A at neutral pH both above and below its main phase temperature (approximately 40-45 degrees C). Analysis of the scattering curves and interpretation of the corresponding thickness distance distribution functions of the lamellar aggregates led to a model in which the lipid A molecules form a bilayer of about 5 nm in thickness. This value for the thickness of the bilayer, as well as the neutron-scattering density profile across the bilayer, can be explained by a molecular model which shows interdigitation of the fatty acid chains of the lipid A.

Three clones from a strain of Salmonella choleraesuis (serogroup C1) were lysogenized with phage 14 (P14) which converts the O-antigen of serogroup C1 salmonellae from O-6,7 to O-6,7,14. The lysogens were compared with their parental non-lysogenic clones with respect to the following properties: average length of O-antigen polysaccharide chains, sensitivity to normal human serum, and mouse-virulence. SDS-polyacrylamide gel electrophoresis of lipopolysaccharides extracted from these bacteria showed that samples from lysogens consisted mainly of long-chained molecules whereas those from non-lysogens contained mainly short-chained molecules. The O-antigen polysaccharide from a lysogen was estimated by chemical analysis to be six times as long as that from a non-lysogen. Lysogens were serum-resistant whereas non-lysogens were serum-sensitive. About 10 times more colony forming units of a lysogen than of a non-lysogen were recovered from the livers and spleens of mice on day 1 and 3 after intraperitoneal inoculation of equal doses. By comparison with S. choleraesuis, lysogenization of S. typhimurium with phage P22 or phage A4 did not affect the chain-length distribution of O-antigen polysaccharide. Our data suggest that phage 14-coded determinants increase efficiency of O-antigen biosynthesis in S. choleraesuis leading to increase in average length of O-polysaccharide chains. Increased serum resistance and mouse virulence are logical consequences of increase in average length of O-polysaccharide chains and represent phage-conferred selective advantage not previously described in Salmonella.

Sixteen monophosphoryl Lipid A (MLA) homologs obtained from the lipopolysaccharides of Salmonella minnesota Re595 were separated by preparative thin layer chromatography into eight fractions. The components of these fractions were analyzed directly (or as structural analogs) and characterized by mass spectrometry. Molecular weights were determined by negative and positive ion fast atom bombardment mass spectrometry and component structures were assigned following a study of fragmentation and metastable ion kinetic energy spectrometry. One fraction (TLC-8) contained a single heptaacyl MLA of Mr = 1,954, a structure previously elucidated (Qureshi, N., Mascagni, P., Ribi, E., and Takayama, K. (1985) J. Biol. Chem. 260, 5271-5278). The remaining seven fractions contained 15 additional MLAs with decreasing acylation. Two of these components have been previously reported in S. minnesota and Salmonella typhimurium. Three of the eight TLC fractions (TLC-8, -7, -6) were found to be biologically active toward human platelets inducing their aggregation and secretion of serotonin. All tested fractions induced varying degrees of phosphorylation of a platelet protein of Mr = 47,000 (P47) reflecting protein kinase C activation (Grabarek, J., Her, G. R., Reinhold, V. N., and Hawiger, J. J. (1990) J. Biol. Chem. 265, 8117-8121).

We previously reported that human blood platelets are directly stimulated by endotoxic Lipid A via the protein kinase C pathway (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82, 964-971). To study the relationship between the molecular structure of Lipid A and its ability to activate human platelets, we used Lipid A homologs derived from Salmonella minnesota Re595 lipopolysaccharide. Preparations of Lipid A are heterogeneous in regard to the degree of substitution of fatty acids which result in multiple homologs. These were separated by thin-layer chromatography and characterized by fast atom bombardment spectroscopy and related techniques (Johnson R. S., Her, G.-R., Grabarek, J., Hawiger, J., and Reinhold, V. N. (1990) J. Biol. Chem. 265, 8108-8116). The homologs of monophosphoryl Lipid A (MLA) present in fractions TLC-8 (heptaacyl MLA ion, m/z 1953), TLC-7 (three hexaacyl species with predominant MLA ion m/z 1715), and TLC-6 (four pentaacyl homologs with predominant MLA ion, m/z 1505) induced secretion of [14C]serotonin and aggregation of platelets. Lipid A homologs in fractions TLC-5 (three tetraacyl MLA ions, m/z 1323, 1307, and 1279), TLC-4 (one major triacyl MLA ion, m/z 1097), TLC-3 (tetraacyl MLA ion, m/z 1278), TLC-2 (a diphosphoryl hexaacyl Lipid A ion, m/z 1795, and several ions of low abundance), and TLC-1 (two ions, m/z 1097 and 666) were not active in regard to human platelet aggregation and [14C]serotonin secretion. The most active homolog was heptaacyl MLA ion, m/z 1953, present in TLC-8, while homologs present in TLC-7 and TLC-6 were 5 and 10 times less active, respectively. Rapid phosphorylation of a human platelet protein of Mr 40,000-47,000 (P47), a substrate for protein kinase C activation, preceded secretion of serotonin when platelets were triggered by the most active heptaacyl MLA ion, m/z 1953. These events were time-dependent, with half-maximal response of phosphorylation of P47 at 30 s and [14C]serotonin secretion at 45 s. A marked difference in the degree of phosphorylation of P47 was observed with heptaacyl MLA homolog present in TLC-8 inducing complete phosphorylation (97%), whereas less acylated Lipid A homologs present in TLC-1 caused marginal phosphorylation (20%). These results indicate that the degree of acylation of monophosphoryl Lipid A determines its functional properties toward human platelets in regard to secretion of [14C]serotonin, aggregation, and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)

Murine monoclonal antibodies (MAbs) and immune rabbit serum were raised against the rough mutant of Salmonella minnesota strain R595. These antibodies were tested for their ability to inhibit LPS-induced B-cell mitogenicity and neutralise LPS toxicity in chick embryos. Immune rabbit serum inhibited both mitogenicity and LPS lethality. None of the MAbs or a cocktail of antibodies were able to neutralise LPS lethality in chick embryos. However, they were able to inhibit mitogenicity by varying degrees.

The relationship between formation of the Mg2(+)-induced hexagonal lattice structure by R-form lipopolysaccharides (LPS) and chemical structure of their R-cores was investigated using different kinds of R-form LPS from a series of mutants of Salmonella minnesota or S. typhimurium. The optimal experimental condition for formation of the hexagonal lattice structure was to suspend LPS preparations, from which cationic material was removed by electrodialysis, in 50 mM tris (hydroxymethyl) aminomethane buffer at pH 8.5 containing 10 mM MgCl2. Under this experimental condition, Rb1 LPS formed the hexagonal lattice structure with the lattice constant of 14.0 +/- 0.2 nm. Ra LPS, which possesses the full length of R-core, also formed the hexagonal lattice structure but its lattice constant was larger (18.1 +/- 0.2 nm) than that of Rb1 LPS (the lattice structure by Ra LPS was looser than that by Rb1 LPS). All the other R-form LPS preparations tested, RcP+, PcP-, Rd1P-, and Re LPS, whose R-cores are shorter than that of Rb1 LPS, did not form the hexagonal lattice structure, but formed membranous structures showing various shapes which consisted of multiple bilayer structures. Failure to form the hexagonal lattice structure was the common feature of these kinds of R-form LPS irrespective of temperature at which the LPS suspensions in 10 mM MgCl2-50 mM Tris buffer were incubated. From the results of the present study it was concluded that capability of R-form LPS to form the hexagonal lattice structure has a close correlation with the chemical structure of their R-cores.

Salmonella madelia reported to express the O-antigenic factors 1, 6, 14, and 25, defined in the Kauffmann-White classification system, was found to produce three different homogeneous lipopolysaccharides, which differed in having three structurally distinct O-polysaccharide components. The O-polysaccharide fraction obtained by mild acetic acid hydrolysis of the S. madelia lipopolysaccharide was analyzed by chemical composition, nitrous acid deamination, periodate oxidation, methylation, and 1H and 13C nuclear magnetic resonance methods and was demonstrated to be composed of three polysaccharides, PS(I), PS(II), and PS(III), which had the structures of repeating oligosaccharide units: (formula; see text)

Salmonella boecker, which belongs to group 0:6, 14(H) and shows the antigenic factors 6, 14, [1], and [25], defined by the Kauffmann-White system, produces two lipopolysaccharides differing from each other in the structures of their 0-poly-saccharide moieties. By glycose composition, partial hydrolysis, nitrous acid deamination, methylation, optical rotation, and 1H and 13C nuclear magnetic resonance studies, the O-polysaccharides were demonstrated to be high-molecular-weight polymers (I and II) composed of either structurally related repeating tetrasaccharide or repeating pentasaccharide units having the structures and (table; see text).

The O-haptenic polysaccharide of Salmonella montevideo has been reported to contain glyceraldehyde at its reducing terminus. However, O-hapten preparations from a pmi galE mutant contained products of partial hydrolysis of lipopolysaccharide, which in separate experiments gave [3H]glycerol upon treatment with perchloric acid and [3H]aBH4. Further study of the O-hapten reducing terminus suggested that it was actually mannose.

The paper describes cultivations of 4 Salmonella S-forms and 1 SR mutant, performed in complex medium under constant conditions of temperature, pH and aeration. The experiments show that lipopolysaccharide (LPS) biosynthesis underlies quantitative differences with the growth phases, resulting in changes in the LPS content of the cell masses. During the exponential phase a decline takes place in the percentage of LPS contained by the 4 S-forms. In addition, in the phase of delayed growth acceleration, 3 of these strains exhibit temporary, complete stagnation in LPS formation. When the cultures enter the stationary phase, LPS biosynthesis also discontinues. The SR-mutant differs from the S-forms especially in that the rate of LPS synthesis and with it, the percent lipopolysaccharide content of the cells, increase greatly in the exponential growth phase. The causes and effects of the changes observed are discussed.

The O-specific polysaccharide of Salmonella arizonae O59 (Arizona 19) is composed of D-galactose, N-acetyl-D-glucosamine, and N-acetyl-L-fucosamine (FucNAc, 2-acetamido-2,6-dideoxy-L-galactose) in the ratio 1:1:1. The computerized calculation of the 13C NMR spectrum of the polysaccharide, based on the monosaccharide composition, spectra of the free monosaccharides and glycosydation effects, together with the chemical analysis (methylation and Smith degradation) showed that the polysaccharide is built up of trisaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp(1----3)-beta-D-GlcNAcp-(1----2)-beta- D-Galp-1(----. The molecular basis of serological interrelations between S. arizonae O59 and Pseudomonas aeruginosa O7 (Lányi) is discussed.

The anomeric configurations of the reducing terminal glucosamine and 4-amino-4-deoxy-L-arabinose phosphates in lipopolysaccharide from Salmonella minnesota R595 have been determined by nuclear magnetic resonance. Chemical shifts for the anomeric protons were obtained by selective decoupling of the phosphorus spectrum and proton-proton coupling constants by polarization transfer from protons to phosphorus. In both cases, the phosphate is attached to the sugar in an axial orientation.

Lipopolysaccharides are integral components of the outer membrane of Gram-negative bacteria and they participate in various membrane functions essential for bacterial growth and survival. Lipopolysaccharides also represent the endotoxins of Gram-negative bacteria and possibly play a role for the pathogenesis and manifestations of bacterial infections. These biological activities are mediated mainly by the lipid component of lipopolysaccharides, termed lipid A. Chemically, lipid A consists of a beta 1, 6-linked D-glucosamine disaccharide which carries substituted phosphoryl groups and a range of D-3-hydroxy and D-3-acyloxyacyl residues, the latter being arranged in a hexagonal dense packing. A number of experimental data allow the conclusion that the highly ordered and compact lipid A structure confers stability to the outer membrane, renders it less permeable to lipophilic molecules and by providing a proper fluidity stabilizes the conformation of biologically active membrane proteins. For endotoxic activities of lipid A the polar substituents of phosphate residues are dispensable. The presence of D-3-hydroxy (or acyloxy) acyl-groups, linked to the glucosamine disaccharide, however, seems to be of importance. Analyses of now available synthetic lipid A analogues are expected to allow a more precise characterization of substructures and conformations required for the expression of physiological functions and endotoxic activities of lipid A.

The polymorphism of lipid A, the endotoxic principle of the lipopolysaccharides of gram-negative bacteria, has been investigated in the fully hydrated state at temperatures between 5 degrees and 58 degrees C via Fourier-transform infrared spectroscopy. These measurements were supplemented by X-ray diffraction, fluorescence intensity techniques and differential thermal analysis. Up to three distinct phase transitions could be detected, with the main transition temperatures lying at approximately 41 degrees, 46 degrees, 44 degrees and 47 degrees C for Escherichia coli lipid A, Salmonella minnesota lipid A, and the synthetic lipid A compounds 506 and 516, respectively. 4'-Monophosphoryl-lipid A samples exhibited their main transition temperatures at considerably higher temperatures (about 52 degrees C for E. coli lipid A). The analysis of greater than CH2 stretching absorption bands as well as the wide-angle scattering behaviour of the lipid A samples showed that the main transition apparently involved the completion of hydrocarbon chain melting of lipid A, as typically observed for phospholipids. However, the phase transition behaviour was found to be much more complex than that usually observed for model phospholipid systems. Even below the main transition temperature, considerable amounts of the methylene segments of the acyl chains of lipid A were found to assume gauche conformations. These conformational changes might be related to the occurrence of up to two further transitions located at about 22 degrees, 30 degrees, 27 degrees and 25.5 degrees C (first transition) and at about 34 degrees, 42 degrees, 38.5 degrees and 40.5 degrees C (second transition) for E. coli lipid A, S. minnesota lipid A and the synthetic lipid A compounds 506 and 516, respectively. Furthermore, by the analysis of some characteristic infrared absorption bands related to the hydrophilic backbone, it could be demonstrated that the temperature-induced conformational changes occurring within the hydrocarbon chains were constantly and simultaneously accompanied by detectable rearrangements within the interfacial region and the polar head group of lipid A. The following conclusions were drawn: Up to about 30 degrees C the lipid A assemblies were supposed to adopt virtually bilayered, true lamellar arrangements, as revealed by the analysis of greater than CH2 scissoring vibrations and X-ray diffraction pattern. However, as indicated by fluorometric techniques, no stable closed vesicles seemed to be formed even under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

The smooth lipopolysaccharide produced by Salmonella eimsbuttel, which had the O:6, O:7, and O:14 antigenic factors defined in the Kauffmann-White classification, was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, composition analysis, methylation, periodate oxidation, deamination, and 1H and 13C nuclear magnetic resonance studies to contain a high molecular weight O-chain polysaccharide composed of D-mannose (four parts), D-glucose (one part), and 2-acetamido-2-deoxy-D-glucose (one part). It was a branched polymer of a repeating hexasaccharide unit having the structure (formula; see text).

Salmonella minnesota R 595 (Re) and other Salmonella strains incorporate cis-delta 9-16:1 (palmitoleic acid) at the expense of mainly dodecanoic acid into the lipid-A portion of lipopolysaccharides, when the cells are grown at low temperature (12 degrees C). It has recently been shown, that in S. minnesota R 595 grown at 37 degrees C dodecanoic acid is linked to the 3-hydroxyl group of an amide-bound 3-hydroxytetradecanoic acid. The present data suggest, that cis-delta 9-16:1 occupies the same position in lipid A (12 degrees C).

The morphological heterogeneity of lipopolysaccharides (LPSs) among salmonella mutants with different LPS chemotypes was analyzed in silver-stained polyacrylamide gels. The biochemical differences in the LPS chemotypes were reflected in the unique profiles of the purified LPSs. The LPS profiles in the whole-cell lysates were also unique for each chemotype. (Whole-cell lysates were assessed by a method which preferentially silver stains LPS and by a proteinase K digest of whole-cell lysates. The silver-stained LPS profiles of proteinase K-digested lysates were similar to the homologous purified LPS and could be used to preliminarily characterize the LPS chemotype before purification.) In summary, biochemical variation in LPS composition can be detected in silver-stained polyacrylamide gels.

The lipopolysaccharide produced by Salmonella livingstone (O:6,7) was composed of an antigenic O-polysaccharide which was shown by composition, methylation analysis, and high resolution nuclear magnetic resonance studies to be a high molecular weight polymer containing D-glucose, 2-acetamido-2-deoxy-D-glucose, and D-mannose residues (1:1:4) composed in a repeating hexasaccharide unit having the structure: (formula; see text)

In group C1 salmonellae, rfe and rff genes linked to the ilv locus specify the synthesis of a glycolipid called the enterobacterial common antigen. In contrast, in group B salmonellae the synthesis requires in addition some of the genes in the rfb cluster, the main genetic determinant of the O side chains of lipopolysaccharide. In an effort to define the biochemical functions of these rfb genes, we looked in Salmonella typhimurium LT2 (group B) for rfb mutants in which the synthesis of both enterobacterial common antigen and the O side chains would be blocked in a manner suppressible by the wild-type rfe cluster of S. montevideo, of group C1. We found one mutant with these characteristics. This rfb mutation affected the activity of dTDPglucose pyrophosphorylase (glucose-1-phosphate thymidylyltransferase, EC 2.7.7.24). Whereas the rfe cluster of S. montevideo contained a gene producing this enzyme activity, there was no evidence for the presence of such a gene in the rfe cluster of group B strains. These results also showed that the synthesis of dTDP-glucose is necessary for the biosynthesis of enterobacterial common antigen; this conclusion fits with the recent demonstration of 4-acetamido-4,6-dideoxy-D-galactose as a component of enterobacterial common antigen (Lugowski et al., Carbohydr. Res. 118:173-181, 1983), because the biosynthesis of the donor of this sugar, dTDP-4-acetamido-4,6-dideoxy-D-galactose, requires dTDPglucose pyrophosphorylase.

Lipopolysaccharides of Salmonella minnesota rough mutants were treated with 20 mM acetate buffer pH 4.4 at 70 degrees C/3 h. After dialysis of the hydrolysates about one third of the total 3-deoxy-D-mannooctulosonic acid (dOclA) content but no neutral sugars were found in the dialysate. By high-voltage paper electrophoresis, a compound with the mobility of 1.2 relative to dOclA could be isolated from the dialysate. It was identified as a dOclA disaccharide by hydrolysis without or after reduction with sodium borohydride and by analysis with the thiobarbituric acid assay under different conditions. The ketosidic linkage in the disaccharide is assumed to be 2.4 or 2.5.

Lipopolysaccharides from different R mutants of Salmonella minnesota and Salmonella typhimurium belonging to chemotypes Ra to Re, as well as from three SR mutants of Salmonella typhimurium were selected for a study of their precipitability with Concanavalin A. Predictions as to the outcome of the reaction could be made since both the chemical structure of the Salmonella R lipopolysaccharides and structural requirements for a positive reaction with Concanavalin A are well established. Precipitation studies in the immuno-electrophoretic assay and in the microcapillary test were carried out with alkali-treated lipopolysaccharides as untreated lipopolysaccharide is too highly aggregated to allow a sufficient migration in agarose layers. Lipopolysaccharides of all mutants--except the SR mutants--were obtained by the phenol/chloroform/petroleum ether method in order to avoid contaminations by glucans or glycogen which are known to occur in phenol/water extracted lipopolysaccharides and which would lead to erroneous results. Additional precipitation studies were carried out with two other lectins of different polysaccharide specificity: Wheat Germ Agglutinin and Soybean Agglutinin. As expected, lipopolysaccharides of chemotypes Ra, Rb1, and RcP- mutants reacted strongly with Concanavalin A, whereas no reaction was demonstrable with lipopolysaccharides of chemotypes Rb2, Rb3, Rd and Re mutants. The lipopolysaccharide of an RcP+ mutant unexpectedly failed to precipitate unless it was dephosphorylated with HF. This artificially prepared RcP-lipopolysaccharide showed a strong reaction, thus demonstrating that negative charges in the direct neighborhood of reactive sugar units as in RcP+ LPS may prevent precipitation with Concanavalin A. No reactivity demonstrable by precipitation could be obtained using either Wheat Germ Agglutinin or Soybean Agglutinin with alkali-treated lipopolysaccharide even of those chemotypes which had the supposedly reactive sugar in a terminal position, such as N-acetyl-D-glucosamine in Ra mutants (Wheat Germ Agglutinin) or D-galactose in Rb2 or Rb3 mutants (Soybean Agglutinin).

Chemical and serological investigations were carried out on lipopolysaccharides of 4 Salmonella S-forms and of 1 SR-mutant, extracted from bacteria at different ages of culture (early exponential to stationary growth phase). The results show that the fatty acid composition of Lipid A (lauric-, myristic-, palmitic-, and beta-hydroxy-myristic acids) does not undergo any significant change during the growth of the cultures. However, there are differences in the molar ratios of the fatty acids from strain to strain. In all phases of growth Lipid A is substituted by basaloligosaccharide, to the same extent, as can be seen from the constant ratios of beta-hydroxy-myristic acid: heptose. Serological experiments (haemagglutination inhibition tests, absorption of antibodies by LPS-coated erythrocytes) showed that in no case the basaloligosaccharide is completely substituted by O-specific chains and that basaloligosaccharide exhibits free R-antigen structures which are mainly of chemotypes Ra, Rb and Rc, for the SR-mutant only of types Ra and Rb. There is no demonstrable dependence upon the phases of growth. In the O-specific polysaccharide chains the sugars of the main chain and the side bound dideoxy sugars (abequose and tyvelose) show a constant 1:1 molar ratio in all phases. In the case of S. typhimurium, antigen factors 1, 4 and 12(2), the biosynthesis of which is controlled by modifying oaf genes and/or by a lysogenic phage, are of a somewhat weaker expression in the exponential phase than in the latter phases of growth. In the SR-mutant, lipopolysaccarides with (low) serological O1 and O12(2) activity are only extractable by the phenol/water method, but not by the PCP method. In three out of four S-forms, changes occur in the length of the O-specific polysaccharide chains, whereas the number of repeating units of the fourth strain remains almost unchanged. The lipopolysaccharides of the SR-mutant contain in all phases of growth about one repeating unit. In all strains the covering of the cell surface by lipopolysaccharide molecules changes during the course of growth, as can be seen by comparing the relative cell surface and the content of Lipid A fatty acids of the bacteria. Lipid A synthesis in the 4 S-forms is reduced in the exponential phase and/or in the phase of delayed growth acceleration. The extent of biosynthesis of the carbohydrate moiety of lipopolysaccharides is independent of that of Lipoid A. In the SR-mutant, Lipoid A and Polysaccharide are formed in increased amounts in the exponential growth phase.

Rough mutants from Salmonella typhimurium and Salmonella minnesota were transformed with a plasmid containing a 6.5-kilobase insert of DNA from Chlamydia trachomatis assumed to encode a glycosyltransferase. Transformation resulted in the expression of a genus-specific chlamydial epitope on the lipopolysaccharide (LPS) of the recombinant strains. Proteinase K-digested whole-cell lysates of the recombinants and of controls were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining or Western blot analysis. Two LPS populations were detected in the recombinants, the parent LPS and a faster-migrating component. The latter stained with monoclonal antibody against the genus-specific chlamydial epitope and was not seen in the controls. LPS was extracted and purified from recombinants of S. minnesota R595 and R4 and characterized by the passive hemolysis and passive hemolysis inhibition assays and by hydrolysis kinetics. Different antigenic determinants could be distinguished from each other by the passive hemolysis inhibition test with monospecific antigen-antibody reactions. Rabbits were immunized with heat-killed recombinant bacteria to study the immunogenic properties of the recombinants. In all animals, antibodies were raised against the parent core specificity and against the chlamydia-specific epitope. The data show that the recombinant bacteria are useful as immunogens to prepare polyclonal antisera against chlamydiae and that LPS isolated from them exhibits the same antigenic determinants as chlamydial LPS and may thus be used as a substitute for chlamydial LPS in serological assays.

Exposure to endotoxin has been associated with systemic toxicity, including pulmonary disorders such as byssinosis, as well as with beneficial biologic activities such as adjuvanticity and mitogenicity. The purified lipopolysaccharide (LPS) from endotoxin has been employed to investigate structure-activity relationships for various biologic effects. The current study was undertaken to examine the relationship between LPS structure and its ability to cause respiratory toxicity in guinea pigs after inhalation exposure. Animals were exposed to atmospheres containing 0.076 to 2.1 micrograms/m3 Salmonella minnesota LPS (S. minn. LPS), LPS from the mutant S. minn. Re595, S. minn. Re595 lipid A, and monophosphoryl S. minn. Re595 lipid A (S. minn. Re595 MPL). Each of the LPS aerosols caused increased breathing frequency (f), decreased tidal volume (VT), and airflow disturbance when measured 18 h after the 6-h inhalation exposure. The LPS preparations had equivalent toxicity, whereas the lipid A aerosol had slightly reduced toxicity. The MPL preparation did not produce this respiratory toxicity response. The results indicated that absence of the terminal phosphate group from the reducing end of the lipid A disaccharide destroyed its ability to cause the respiratory effect. These results initiate structure-activity studies of defined LPS in the lung and indicate the possibility of chemically treating endotoxins to remove adverse pulmonary effects.

The paper describes the preparation of the lipopolysaccharide from Salmonella abortus equi as obtained by standardized methods. The include the extraction with pehnol/water followed by phenol/chloroform/petroleum ether extraction, ultra-centrifugation, electrodialysis and conversion to the uniform sodium salt form. Chemical composition and physico chemical properties are described. The preparation, which is free from contaminants, was tested for local Shwartzman reactivity, pyrogenicity, lethal toxicity, mitogenicity, reactivity towards complement and tumoricidal action.

The comparative study of the fatty acid composition of bacterial lipopolysaccharides (LPS) in the genera Escherichia, Shigella, Salmonella has been carried out under identical experimental conditions. The LPS of the bacteria under study have been found to contain a number of saturated fatty acids and oxyacids which could not be previously detected in these bacteria in other studies. In all bacterial strains under study LPS include mainly 3-oxytetradecanoic, tetradecanoic and dodecanoic fatty acids. The essential feature of the fatty acid composition of Salmonella is the presence of 2-oxytetradecanoic acid; this acid is absent in Escherichia and Shigella, which can thus be used as a differentiating criterion. The content of other fatty acids in Salmonella is similar to that in Escherichia and Shigella. These data confirm that the genera Escherichia, Shigella and Salmonella are phylogenetically related, the relationship between Escherichia and Shigella being more close.

Prolonged culturing in the laboratory has resulted in the formation of a stable derivative of the smooth Group E bacterial strain, Salmonella anatum A1, that is sensitive to both the R-core-specific bacteriophage Felix 01 and O-polysaccharide-specific bacteriophage epsilon 15. The variant strain, designated S. anatum A1-1, exhibits a normal number of irreversible binding sites for epsilon 15 but the relative quality and/or accessibility of those sites appears to be diminished. Infectious epsilon 15 phage particles are released more rapidly from S. anatum A1-1 than from its parent under acidic pH conditions known to interfere with the phage DNA ejection step. The purified lipopolysaccharide (LPS) of S. anatum A1-1 exhibits a reduced rhamnose/heptose ratio in chemical assays. Fractionation of this LPS on SDS-urea-polyacrylamide gels followed by silver staining reveals a narrower range of O-polysaccharide chain lengths relative to that of the parent (0 to 20 vs. 0 to 40 repeating units, respectively).

Latex particles sensitized with a Salmonella O-9 monoclonal antibody were used to detect group D salmonellae in blood culture broths. The system was found to be 100% accurate when used on 104 clinical specimens that included 13 S. typhi isolates. Enteric fever could thus be diagnosed as early as 1 day after the initiation of culture in some (18%) cases. The latex particles were also used in an experimental situation to detect soluble S. typhi lipopolysaccharide by a tube agglutination method. This was found to be more than four times as sensitive as the conventional slide agglutination method because at least 50 ng of antigen per ml in either buffer or urine was detected, albeit with less efficiency when serum was used.

Lipopolysaccharide (LPS) of Salmonella dublin, S. enteritidis, S. typhimurium, S. choleraesuis and their derivative strains was analysed to investigate the correlation between LPS and virulence plasmid of Salmonella. All wild-type strains had smooth type LPS, i.e. LPS with long O-specific polysaccharide. The virulence plasmid-cured strain of S. dublin, C524, exhibited a shorter O-specific chain than its parent strain, 5240. No distinct ladder bands were observed at the high molecular weight region on the SDS-PAGE gel for C524 LPS. By chemical analysis the number of O-repeating unit of C524 LPS was shown to be approximately one. The chain length of O-specific polysaccharide was restored by reintroduction of the virulence plasmid. The alteration of LPS by curing and reintroduction of the virulence plasmid was not observed when other wild-type strains of S. dublin were used. In the case of S. enteritidis, S. typhimurium, and S. choleraesuis, alteration of neither chemical composition nor electrophoretical profile of LPS was detected by curing and reintroduction of the virulence plasmids. Those results suggest that certain factor for regulation of the chain length of O-specific polysaccharide is encoded on the virulence plasmid of S. dublin.

The monophosphoryl lipid A (MLA) obtained from the lipopolysaccharides of Salmonella minnesota R595 was fractionated on a silicic acid column to yield the heptaacyl, hexaacyl, and pentaacyl MLA. Each of these MLAs was methylated with diazomethane to yield the dimethyl derivative and purified to homogeneity by reverse-phase high performance liquid chromatography. The molecular ions obtained by positive ion fast atom bombardment mass spectrometry of purified dimethyl heptaacyl MLA allowed us to establish the molecular formula and Mr of C112H211N2O23P and 1983.3, respectively. Cleavage at the glycosidic linkage yielded an oxonium ion of mass 1115, which showed that the distal sugar unit contained one phosphate (dimethyl), two hydroxymyristates, one laurate, and one myristate, while the reducing sugar unit contained two hydroxymyristates and one palmitate. By utilizing two-dimensional NMR spectroscopy, we were able to assign all of the protons of dimethyl heptaacyl MLA. This assignment included the beta protons of the three acyloxyacyl groups. A substantial downfield shift of the protons at the 3- and 3' -carbons was observed, which indicated that these two positions are occupied by ester groups. Fast atom bombardment mass spectral analysis of the hexaacyl and pentaacyl MLAs showed that these structures were identical to the previously designated TLC-3 and TLC-5 fractions, respectively, from Salmonella typhimurium. From this study, the complete structures of the MLA series found in the LPS of S. minnesota can now be described.

We have developed a solid-phase radioimmunoassay technique for specific gram-negative bacterial lipopolysaccharide (LPS) O antigens. The method exploits the high-titer, specific immunoglobulin M response of the rabbit to LPS immunization to measure as little as 5 ng of homologous LPS per ml with less than 0.5% cross-reactivity toward heterologous LPS or culture supernatants. We found that O antigen in complete LPS was less available for antibody binding than O antigen in the soluble polysaccharide derived by mild acid hydrolysis of LPS and that triethylamine-induced disaggregation of complete LPS increased its activity in the assay. Quantitation of O antigen with the assay was thus influenced by the physical state of LPS or "free" O antigen.