Characterization of the cell wall and outer membrane of Rhodopseudomonas capsulata.

Sucrose density gradient centrifugation of cell envelopes of chemotrophically grown cells of Rhodopseudomonas capsulata St. Louis (= ATCC 23782) resulted in the separation of a cytoplasmic membrane from a cell wall fraction (buoyant densities, 1.139 and 1.215 g/cm3, respectively). The cell wall fractions (untreated or Triton extracted) contained peptidoglycan- and lipopolysaccharide-specific components. Their neutral sugar content, mainly rhamnose and galactose, was high (250 and 100 micrograms/mg [dry weight] of material) due to a non-lipopolysaccharide polymer. The fatty acid content was low (less than or equal to 60 micrograms/mg [dry weight] of material), and half of it was contributed by lipopolysaccharide (3-OH-C10:0, C12:1, and 3-oxo-C14:0). The predominant other fatty acid was C18:1. An outer membrane fraction, obtained by lysozyme treatment of the Triton-extracted cell wall, showed essentially the same chemical composition except for almost complete removal of peptidoglycan. Saline extraction (0.9% NaCl, 37 degrees C, 2 h) removed a lipopolysaccharide-protein(-phospholipid?) complex from whole cells of R. capsulata St. Louis. The polypeptide patterns of the cell wall and outer membrane as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comprised 20 to 25 different polypeptides (most of them very faint) and were dominated by a single, heat-modifiable major protein (Mr 69,000 after solubilization below 60 degrees C; Mr 33,000 at temperatures above 70 degrees C).     

Genetic and physical map of the structural genes (nifH,D,K) coding for the nitrogenase complex of Rhodopseudomonas capsulata.

Functional genes coding for the structural components of the nitrogenase complex (nifH,D,K) have been cloned on an 11.8-kilobase-pair HindIII fragment of DNA from the photosynthetic bacterium Rhodopseudomonas capsulata. The genes were physically mapped by hybridization of individual cloned nif genes from Klebsiella pneumoniae and Anabaena sp. strain 7120 to Southern blots of HindIII digests of the cloned R. capsulata fragment, after introduction of HindIII sites into the latter at specified locations by insertion of Tn5. Plasmids with the 11.8-kilobase-pair HindIII fragment containing the Tn5 insertions were also used for complementation tests with chromosomal Nif- mutations and for the generation of subfragments to locate those mutations by marker rescue. The R. capsulata nifH,D,K genes comprise a single unit of expression, with the same organization and polarity as found in K. pneumoniae. However, the R. capsulata nifH,D,K fragment did not complement Nif- point mutations in the corresponding Klebsiella genes, and the Klebsiella nif genes did not function in R. capsulata.     

Mobilization of the genes for photosynthesis from Rhodopseudomonas capsulata by a promiscuous plasmid.

Plasmid pBLM2, a derivative of RP1 with enhanced chromosome mobilization activity in Rhodopseudomonas capsulata, was isolated by screening rare exconjugant clones for sex factor activity. pBLM2 mobilized all known genes affecting photosynthesis as well as chromosomal genes for streptomycin and rifampin resistance and tryptophan and cytochrome biosynthesis. Tight linkage was exhibited among the genes affecting photosynthesis. The frequency of successful transfer of chromosomal markers reached 6 X 10(-4) per donor cell. R-primes were occasionally formed during conjugation, and a number of R-primes bearing the genes for photosynthesis were isolated by screening R. capsulata exconjugants with complementation phenotypes for the ability to transmit plasmid-borne R. capsulata genes to Escherichia coli cells. These R-primes were unstable in R. capsulata, but stable in E. coli or Pseudomonas fluorescens. Complementation and recombination events that occurred upon introduction of R-primes into R. capsulata mutants with altered photosynthetic apparatuses could be visualized as variations in colony pigmentation. Each R-prime studied complemented all known types of mutation affecting the differentiation of the photosynthetic apparatus, and no other R. capsulata gene was identified on those plasmids. The R. capsulata genes borne on the R-primes were not functional in E. coli or P. fluorescens.     

Chemical analysis of and degradation studies on the cell wall lipopolysaccharide of Rhodopseudomonas capsulata

A lipopolysaccharide (LPS) has been isolated from the gram-negative photosynthetic bacterium Rhodopseudomonas capsulata. Chemical analysis revealed the presence of d-glucose, d-galactose, l-rhamnose, 3-O-methyl-l-rhamnose (l-acofriose), d-glucosamine, 2-keto-3-deoxyoctonate, and neuraminic acid. The LPS does not contain l-glycero-d-mannoheptose, a typical component of the LPS of enteric bacteria. Fatty acid analysis showed that, apart from lauric acid, two hydroxy fatty acids (hydroxycaproic and hydroxymyristic acids) are the main components. By hydrolysis in weak acid, the LPS has been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Presumably the lipid A contains a glucosamine backbone. Whereas the OH-groups of glucosamine are esterified with lauric and hydroxycaproic acids, hydroxymyristic acid is linked to the amino group of the sugar. By separation of the degraded polysaccharide by gel filtration, a fraction has been isolated which inhibited hemagglutination in a system containing antiserum, obtained by immunization of rabbits with whole cells, and isolated LPS. This fraction, which includes the determinant group, contains the sugars glucose, rhamnose, and acofriose. A second fraction obtained in this way was found to be serologically inactive and is composed of glucose, galactose, neuraminic acid, and phosphate.     

Possible existence of a novel amphipathic immunostimulator in the phenol-water extracts of Mycobacteriaceae

The extracts having diverse immunostimulating activities were obtained as a water-phase fraction from four bacterial species representing the 4 genera (Mycobacterium, Nocardia, Gordona, and Rhodococcus) of Mycobacteriaceae by the phenol-water method, which is commonly used for extraction of endotoxic lipopolysaccharides (LPS) from gram-negative bacteria and amphipathic substances from gram-positives. These fractions, especially those of G. aurantiaca and R. terrae, showed strong stimulatory effects on murine splenocytes, macrophages of mice and guinea pigs, the immunoadjuvant activities in guinea pigs and mice, and the distinct activities inducing a tumor necrosis factor and interferons alpha/beta and gamma in primed mice. The fractions from G. aurantiaca and R. terrae exhibited potent pyrogenicity and the ability to activate the clotting enzyme cascade of the horseshoe crab (Tachypleus tridentatus). Some of these biological activities were not very different from the potency of the reference endotoxic LPS derived from Escherichia coli or Fusobacterium nucleatum. But the test fractions neither showed the activity to prepare rabbit skin to the local Shwartzman reaction, nor reacted with anti-lipid A conventional and monoclonal antibodies. Furthermore, unlike LPS, these fractions stimulated the splenocytes of C3H/HeJ mice (LPS-Nonresponder). Although the fractions showing the above biological activities have not yet been adequately purified, they contained polysaccharides, whose main constituent sugar is mannose with a smaller amount of arabinose, fatty acids consisting primarily of palmitic, stearic, and tuberculostearic acids, and small amounts of peptides and amino sugars. Since components characteristic of known immunomodulators of bacterial origin, namely endotoxins (lipid A's), cell wall peptidoglycans, lipoteichoic acids, cord factors (trehalose dimycolates), or deoxyribonucleic acids, were practically not detected in these fractions, the agent responsible for the above bioactivities is considered to be a novel substance different from the known, bacterial immunomodulators.     

Enhancement of endotoxicity and reactivity with carbocyanine dye by sonication of lipopolysaccharide

The specificity of endotoxin (lipopolysaccharide, LPS) in the carbocyanine dye reaction was investigated, and then a stoichiometric study of the dye-LPS interaction was conducted with attention to the relationship of biological activities of LPS to the reactivity with the dye. Absorption maxima of some bacterial components in the dye reaction were as follows; LPS from both Escherichia coli and Pseudomonas aeruginosa and lipid A from E. coli LPS, 465 nm; Shigella flexneri LPS, 460 nm; Salmonella minnesota R595 glycolipid, 470 nm; polysaccharide from E. coli LPS, 650 nm; yeast RNA, 620 nm; streptococcal M protein and pyrogenic exotoxin, 610 nm; and free fatty acids, 445-450 nm. The absorbance at 465 nm was increased approximately threefold by sonicating LPS for 1-3 min, which roughly paralleled the decrease in turbidity of the LPS aqueous solution. The Limulus amoebocyte lysate (LAL) gelation activity of LPS increased 10-fold when LPS was sonicated for 0.5-5 min, but it decreased to the control level after further treatment. This decrease, however, was overcome by sonication in the presence of 5 mmol of L-ascorbic acid used as an antioxidant. The LAL gelation activity of LPS was inactivated in parallel with an increase in the ratio (w/w) of dye to LPS from 1.73 to 6.90 in the dye-LPS mixture. Pyrogenicity of LPS was also clearly inactivated when the ratio was over 1.73. The ratios of the height of the beta band at 465 nm (dye-LPS complex) to that of the alpha band at 510 nm (free dye) were increased by sonicating LPS, indicating that the binding character, or stacking tendency, was increased by sonicating LPS.     

Hybridization of cloned Rhodopseudomonas capsulata photosynthesis genes with DNA from other photosynthetic bacteria

The homology of Rhodopseudomonas capsulata DNA segments carrying photosynthesis genes with sequences present in total DNA from certain other photosynthetic and non-photosynthetic bacterial species was determined by hybridization. R. capsulata DNA fragments that carry loci for production of peptide components of the photosynthetic reaction center and light-harvesting I antenna complex were found to hybridize to DNA from some photosynthetic species. However, fragments that carry carotenoid or bacteriochlorophyll biosynthesis genes showed either weak or undetectable heterospecific hybridization under the conditions employed.     

Locus of the Action of Serum and the Role of Lysozyme in the Serum Bactericidal Reaction

The mechanism of the lethal action of human serum on a rough strain of Escherichia coli was investigated by use of serum with and without lysozyme, in medium of low and high osmotic pressure, with cells radioactively labeled in the peptidoglycan polymer, and by electron microscopy. The results suggested that there are two separate components in the bacterial cell wall that afford structural support for the cell. Lysozyme attacked one of these, the peptidoglycan polymer. Serum damaged the other, which is probably the peripherally located lipopolysaccharide-phospholipid complex. The cell wall damage caused by lysozyme-free serum promptly resulted in cell death under usual conditions. In plasmolyzed cells, however, the wall damage was not lethal, presumably because the membrane of the plasmolyzed cell was protected from secondary lethal changes which otherwise occur.     

Photoproduction of h(2) from cellulose by an anaerobic bacterial coculture

Cellulomonas sp. strain ATCC 21399 is a facultatively anaerobic, cellulose-degrading microorganism that does not evolve hydrogen but produces organic acids during cellulose fermentation. Rhodopseudomonas capsulata cannot utilize cellulose, but grows photoheterotrophically under anaerobic conditions on organic acids or sugars. This report describes an anaerobic coculture of the Cellulomonas strain with wild-type R. capsulata or a mutant strain lacking uptake hydrogenase, which photoevolves molecular hydrogen by the nitrogenase system of R. capsulata with cellulose as the sole carbon source. In coculture, the hydrogenase-negative mutant produced 4.6 to 6.2 mol of H(2) per mol of glucose equivalent, compared with 1.2 to 4.3 mol for the wild type.     

Chemical characteristics and endotoxic activity of the lipopolysaccharide of Rahnella aquatilis 2-95

The lipopolysaccharide (LPS) from a new Enterobacteriaceae species, Rahnella aquatilis 2-95, was isolated and investigated. The structural components of the LPS molecule, namely, lipid A, core oligosaccharide, and O-specific polysaccharide, were obtained by mild acid hydrolysis. In lipid A, 3-oxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. The major monosaccharides of the core oligosaccharide were galactose, arabinose, fucose, rhamnose, and an unidentified component. The O-specific polysaccharide was found to be assembled of a repeated trisaccharide unit of the following structure: [structure: see text]. The R. aquatilis 2-95 LPS is less toxic and more pyrogenic as compared to the one from the R. aquatilis 1-95 strain studied earlier. Both acyl and phosphate groups are essential for toxic and pyrogenic activity of R. aquatilis 2-95 LPS.     

Bacterial lipopolysaccharides as inducers of disease resistance in tobacco.

The cell wall component of Pseudomonas solanacearum that induces disease resistance in tobacco was highly heat stable at neutral or alkaline pH but highly labile at acid pH. Activity was unaffected by nucleases and proteases but destroyed by a mixture of beta-glycosidases. Washing of bacterial cell walls released a lipopolysaccharide (LPS) fraction with high inducer activity. Purified LPS, extracted by a variety of procedures from whole cells, isolated cell walls, and culture filtrates of both smooth and rough forms of P. solanacearum, induced disease resistance in tobacco at concentrations as low as 50 microgram/ml. The LPS from the non-plant pathogens Escherichia coli B, E. coli K, and Serratia marcescens was also active. Cell wall protein, free phospholipid, and nucleic acids were not necessary for activity. Moreover, since LPS from rough forms was active, the O-specific polysaccharide of the LPS was not required for activity. Hydrolysis of the remaining core-lipid A linkage or deacylation of lipid A destroyed inducer activity. When injected into tobacco leaves, purified LPS attached to tobacco mesophyll cell walls and induced ultrastructural changes in the host cell similar to those induced by attachment of whole heat-killed bacteria.     

Bacterial lipopolysaccharides as inducers of disease resistance in tobacco.

The cell wall component of Pseudomonas solanacearum that induces disease resistance in tobacco was highly heat stable at neutral or alkaline pH but highly labile at acid pH. Activity was unaffected by nucleases and proteases but destroyed by a mixture of beta-glycosidases. Washing of bacterial cell walls released a lipopolysaccharide (LPS) fraction with high inducer activity. Purified LPS, extracted by a variety of procedures from whole cells, isolated cell walls, and culture filtrates of both smooth and rough forms of P. solanacearum, induced disease resistance in tobacco at concentrations as low as 50 microgram/ml. The LPS from the non-plant pathogens Escherichia coli B, E. coli K, and Serratia marcescens was also active. Cell wall protein, free phospholipid, and nucleic acids were not necessary for activity. Moreover, since LPS from rough forms was active, the O-specific polysaccharide of the LPS was not required for activity. Hydrolysis of the remaining core-lipid A linkage or deacylation of lipid A destroyed inducer activity. When injected into tobacco leaves, purified LPS attached to tobacco mesophyll cell walls and induced ultrastructural changes in the host cell similar to those induced by attachment of whole heat-killed bacteria.     

Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata

A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1. More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length. Mutants deficient in uptake hydrogenase (Hup-) were obtained from R. capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis. The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis. Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay. The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4. The cloned R. capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease. Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10. Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R. capsulata.     

Overexpression of the waaZ gene leads to modification of the structure of the inner core region of Escherichia coli lipopolysaccharide,truncation of the outer core,and reduction of the amount of O polysaccharide on the cell surface

The waa gene cluster is responsible for the biosynthesis of the lipopolysaccharide (LPS) core region in Escherichia coli and Salmonella: Homologs of the waaZ gene product are encoded by the waa gene clusters of Salmonella enterica and E. coli strains with the K-12 and R2 core types. Overexpression of WaaZ in E. coli and S. enterica led to a modified LPS structure showing core truncations and (where relevant) to a reduction in the amount of O-polysaccharide side chains. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the predominant LPS structures in an E. coli isolate with an R1 core (waaZ is lacking from the type R1 waa gene cluster) with a copy of the waaZ gene added on a plasmid. Novel truncated LPS structures, lacking up to 3 hexoses from the outer core, resulted from WaaZ overexpression. The truncated molecules also contained a KdoIII residue not normally found in the R1 core.     

Effect of oxygen on acetylene reduction by photosynthetic bacteria

The effect of dissolved oxygen concentration on nitrogenase activity was studied in three species of photosynthetic bacteria. The O2 concentration in the cell suspension was measured with an O2 electrode inserted into the reaction vessel. Acetylene reduction by whole cells of Rhodopseudomonas capsulata, Rhodospirillum rubrum, and Chromatium vinosum strain D was inhibited 50% by 0.73, 0.32, and 0.26 microM O2, respectively. The inhibition of the activity by O2 in R. capsulata usually was reversed completely by reestablishing anaerobic conditions. In R. rubrum and C. vinosum the inhibition was only partially reversible. The respiration rate of R. capsulata was the highest of the three, that of R. rubrum was intermediate, and that of C. vinosum was lowest. R. capsulata and R. rubrum cells were broken after their acetylene reduction activity in vivo had been completely inhibited by O2, and nitrogenase was found to be active in vitro. A concentration of cyanide that did not affect acetylene reduction activity, but which inhibited 75 to 90% of the O2 uptake by whole cells of R. capsulata, shifted the O2 concentration causing 50% inhibition of nitrogenase activity from 0.73 microM to 2.03 microM. These results are in accordance with the assumption that within a limited range of O2 concentrations, the respiratory activity of the cells is enough to scavenge the O2 and to keep the interior of the cells essentially anaerobic. It is suggested that O2 inhibits nitrogenase activity by competing for a limited supply of electrons. When cyanide is present, respiration is slower but is adequate to keep the nitrogenase environment in the cell anaerobic. The lower respiration rate may allow a greater proportion of the electrons to be used for acetylene reduction.     

Comparative study of electrokinetic potentials and binding affinity of lipopolysaccharides-chitosan complexes

Electrokinetic properties of complexes of chitosan (Ch) with lipopolysaccharides (LPSs) from Escherichia coli O55:B5, Yersinia pseudotuberculosis 1B 598, and Proteus vulgaris O25 (48/57) and their size distribution were investigated using zeta-potential distribution assay and quasi-elastic light scattering. The interaction of LPS from different microorganisms with chitosan at the same w/w ratio of components (1:1) resulted in the formation of complexes in which the negative charge of LPS was neutralized (LPS from E. coli) or overcompensated (Y. pseudotuberculosis and P. vulgaris). The changing in size of the endotoxin aggregates during binding with chitosan was observed. The binding constants of chitosan with LPSs were determined by a method with using the anionic dye Orange II. The LPS from E. coli possess higher affinity to chitosan in comparison with the two others samples of endotoxin.     

Physiology of dark fermentative growth of Rhodopseudomonas capsulata

The photosynthetic bacterium Rhodopseudomonas capsulata can grow under anaerobic conditions with light as the energy source or, alternatively, in darkness with D-fructose or certain other sugars as the sole source of carbon and energy. Growth in the latter mode requires an "accessory oxidant" such as trimethylamine-N-oxide, and the resulting cells contain the photosynthetic pigments characteristic of R. capsulata (associated with intracytoplasmic membranes) and substantial deposits of poly-beta-hydroxybutyrate. In dark anaerobic batch cultures in fructose plus trimethylamine-N-oxide medium, trimethylamine formation parallels growth, and typical fermentation products accumulate, namely, CO2 and formic, acetic, and lactic acids. These products are also found in dark anaerobic continuous cultures of R. capsulata; acetic acid and CO2 predominate when fructose is limiting, whereas formic and lactic acids are observed at elevated concentrations when trimethylamine-N-oxide is the limiting nutrient. Evidence is presented to support the conclusions that ATP generation during anaerobic dark growth of R. capsulata on fructose plus trimethylamine-N-oxide occurs by substrate level phosphorylations associated with classical glycolysis and pyruvate dissimilation, and that the required accessory oxidant functions as an electron sink to permit the management of fermentative redox balance, rather than as a terminal electron acceptor necessary for electron transport-driven phosphorylation.     

Deacylation of lipopolysaccharide in whole Escherichia coli during destruction by cellular and extracellular components of a rabbit peritoneal inflammatory exudate

Deacylation of purified lipopolysaccharides (LPS) markedly reduces its toxicity toward mammals. However, the biological significance of LPS deacylation during infection of the mammalian host is uncertain, particularly because the ability of acyloxyacyl hydrolase, the leukocyte enzyme that deacylates purified LPS, to attack LPS residing in the bacterial cell envelope has not been established. We recently showed that the cellular and extracellular components of a rabbit sterile inflammatory exudate are capable of extensive and selective removal of secondary acyl chains from purified LPS. We now report that LPS as a constituent of the bacterial envelope is also subject to deacylation in the same inflammatory setting. Using Escherichia coli LCD25, a strain that exclusively incorporates radiolabeled acetate into fatty acids, we quantitated LPS deacylation as the loss of radiolabeled secondary (laurate and myristate) and primary fatty acids (3-hydroxymyristate) from the LPS backbone. Isolated mononuclear cells and neutrophils removed 50% and 20-30%, respectively, of the secondary acyl chains of the LPS of ingested whole bacteria. When bacteria were killed extracellularly during incubation with ascitic fluid, no LPS deacylation occurred. In this setting, the addition of neutrophils had no effect, but addition of mononuclear cells resulted in removal of >40% of the secondary acyl chains by 20 h. Deacylation of LPS was always restricted to the secondary acyl chains. Thus, in an inflammatory exudate, primarily in mononuclear phagocytes, the LPS in whole bacteria undergoes substantial and selective acyloxyacyl hydrolase-like deacylation, both after phagocytosis of intact bacteria and after uptake of LPS shed from extracellularly killed bacteria. This study demonstrates for the first time that the destruction of Gram-negative bacteria by a mammalian host is not restricted to degradation of phospholipids, protein, and RNA, but also includes extensive deacylation of the envelope LPS.     

Chemical characterization of lipopolysaccharide from Edwardsiella ictaluri, a fish pathogen

The chemical components of lipopolysaccharide (LPS) from the fish pathogen Edwardsiella ictaluri (Ed. ictaluri) were analyzed by SDS-PAGE, gas chromatography, and spectrophotometry, and compared with those of Salmonella typhimurium and Escherichia coli 0111:B4. Only four to five low molecular weight species of LPS from Ed. ictaluri were detected by silver staining after separation by polyacrylamide gel electrophoresis. The low molecular weight species, as well as a low sugar content, indicate that the LPS from Ed. ictaluri was of the rough type, compared with that of S. typhimurium and E. coli which were both of the smooth type LPS. Quantitatively, mannose was not a major sugar component in Ed. ictaluri, unlike S. typhimurium. Palmitic, palmitoleic, and cis-9,10-methylene-hexadecanoic acids were predominant fatty acids among the total cellular lipids of Ed. ictaluri. C14 fatty acids comprised 78% of the total in the LPS of this bacterium, with beta-hydroxy-myristate representing 55%. The results of this study suggest that the lipid A segment of the LPS molecule of Ed. ictaluri is similar to S. typhimurium and E. coli, at least with respect to fatty acid content; however, the core polysaccharide of E. ictaluri differs in that it has twice the heptose content.     

Lipopolysaccharide containing L-acofriose in the filamentous blue-green alga Anabaena variabilis

For the first time, an O-antigenic lipopolysaccharide (LPS) has been isolated from a filamentous blue-green alga (Anabaena variabilis). It was extractable with phenol-water, resulting in extraction of the bulk of the LPS into the phenol phase. The polysaccharide moiety of this LPS consists of l-rhamnose, its 3-O-methyl ether l-acofriose, d-mannose, d-glucose, and d-galactose. l-Glycero-d-mannoheptose and 2-keto-3-deoxyoctonate, the two characteristic sugar components of enteric LPS, and phosphate groups are absent from the A. variabilis O antigen. The only amino sugar present is d-glucosamine. Three hydroxy fatty acids were identified, namely, beta-hydroxymyristic, beta-hydroxypalmitic and beta-hydroxystearic acids, in addition to palmitic and unidentified fatty acid. The LPS of A. variabilis is localized in the outermost cell wall layer and behaves like a bacterial O antigen in serological tests. The passive hemagglutination yielded high titers with isolated LPS (pretreated by heat or by alkali) and rabbit antisera prepared against living or heat-killed cells. The position of the precipitation arcs after immunoelectrophoresis of the O antigen indicates the lack of charged groups. The water phase of the phenol-water extract contains, in high yield, a glucose polymer. It is serologically inactive as shown by the passive hemagglutination test and by agar-gel precipitation.