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Epidemiological data and animal studies suggest that helminth infection exerts potent immunomodulatory effects that dampen host immunity against unrelated pathogens. Despite this notion, we unexpectedly discovered that prior helminth infection resulted in enhanced protection against subsequent systemic and enteric bacterial infection. A population of virtual memory CD8 T (CD8 TVM) cells underwent marked expansion upon infection with the helminth Heligmosomoides polygurusby an IL-4-regulated, antigen-independent mechanism. CD8 TVMcells disseminated to secondary lymphoid organs and established a major population of the systemic CD8 T cell pool. IL-4 production elicited by protein immunization or selective activation of natural killer T cells also results in the expansion of CD8 TVMcells. Notably, CD8 TVMcells expanded by helminth infection are sufficient to transfer innate non-cognate protection against bacteria to naïve animals. This innate non-cognate “collateral protection” mediated by CD8 TVMmight provide parasitized animals an advantage against subsequent unrelated infections, and represents a potential novel strategy for vaccination.

Homeostasis is a fundamental principle of biological systems. A paradigm of immune homeostasis is the remarkably constant number of naive T and B lymphocytes in the body that continuously circulate through the secondary lymphoid organs to maximize immune surveillance. Whether the dynamics and distribution of the systemic naive lymphocyte pool is affected following organ-specific infection is not known. Here we show that, following infection of mice with an enteric helminth, naive T and B lymphocytes accumulate in the T helper type 2-reactive mesenteric lymph node while they are concurrently depleted from non-draining peripheral lymph nodes. This systemic redistribution of naive lymphocytes is sustained into the chronic phase of the infection, requires lymphotoxin beta receptor-dependent signals and is associated with a reduced ability of parasitized animals to mount antigen-specific cellular and humoral immune responses to heterologous immunization or infection at peripheral sites. Our data suggest that the function of the homeostatic naive lymphocyte pool can be modulated by its systemic distribution following infection and may provide a novel concept underlying compromised immune responsiveness at peripheral sites in helminth-infected individuals.

Capnocytophaga spp. contain a group of unusual sulfonolipids, called capnoids (W. Godchaux III and E. R. Leadbetter, J. Bacteriol. 144:592-602, 1980). One of these lipids, capnine, is 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid; the others are, apparently, N-acylated versions of capnine. The lipids were found, in amounts ranging from 2.5 to 16 mumol of capnoid sulfur per g of cells (wet weight), in two Cytophaga spp. and also in several closely related organisms: several Capnocytophaga spp., Sporocytophaga myxococcoides, two Flexibacter spp., and two Flavobacterium spp. With the exception of the flavobacteria, all of these bacteria have been shown to exhibit gliding motility. The two Flavobacterium spp. belong to a subset of that genus that shares many other characteristics with the cytophagas. Only the Capnocytophaga spp. contained large quantities of capnine as such; in all of the others, most (and possibly all) of the capnoids were present as N-acylcapnines. Capnoid-negative bacteria included some gliding organisms that may not be closely related to the cytophagas: two fruiting myxobacters, a gliding cyanobacterium (Plectonema sp.), Beggiatoa alba, Vitreoscilla stercoraria, Herpetosiphon aurantiacus, and Lysobacter enzymogenes. Nongliding bacteria representing nine genera were also tested, and all of these fell into the capnoid-negative group.

The assimilatory nitrate reductase of the N2-fixing bacterium Azotobacter chroococcum has been prepared in a soluble form from cells grown with nitrate as the nitrogen source, and some of its properties (electron donors and cofactors, Kmvalues for substrates, molecular weight, inhibitors, activators, etc.) have been studied. The enzyme is of an inducible nature and can exist in two interconvertible forms, either active or inactive.

A group of unusual sulfonolipids was found in bacteria of the genus Capnocytophaga. One of these lipids, to which we have assigned the trivial name capnine, was isolated in 98% pure form and was identified, by infrared absorption spectrometry, high-resolution mass spectrometry, and other methods, as 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid. Another lipid appears to be an N-acylated version of capnine; after acid hydrolysis, its sulfur was recovered in a form chromatographically indistinguishable from that of capnine. The new lipids are related structurally to sphingosine and the ceramides, respectively, but differ markedly from those compounds in important respects, notably the presence of the sulfonate group. Some Capnocytophaga strains accumulated mostly capnine, whereas others accumulated mostly N-acylcapnine. All seven strains examined were found to contain the new lipids, in amounts ranging from 7 to 16 mumol/g of cells (wet weight). The lipids were found in isolated cell envelopes, where they were present in amounts ranging up to 400 mg/g of envelope protein; they are, accordingly, major cell components.

Earlier (Godchaux, W., and Leadbetter, E. R. (1980) J. Bacteriol. 144, 592-602; (1983) J. Bacteriol. 153, 1238-1246) we demonstrated that an unusual class of sulfonolipids are major components of the cell envelope of gliding bacteria of the genus Cytophaga and of closely related genera. One of these lipids, to which we have assigned the trivial name capnine, was purified and was shown to be 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid (which might also be named as 1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Though capnine accumulates as such in the cells of some Capnocytophaga spp., most organisms of the Cytophaga-like genera contain, instead, sulfonolipids that are less polar than capnine. These less polar lipids have been purified from a Capnocytophaga sp., a marine Cytophaga sp., Cytophaga johnsonae, and a Flexibacter sp. Acid methanolysis of the lipids yielded both aminosulfonates and a collection of fatty acid methyl esters. The infrared absorption spectra of the lipids indicated that the fatty acids were in amide (and not ester) linkage to the aminosulfonates. In every instance, analysis by mass spectrometry and other methods revealed that most, if not all, of the aminosulfonates obtained by methanolysis were structurally identical to capnine (though small amounts of variants of that compound may be present in some cases). The less polar sulfonolipids are, therefore, predominantly N-fatty acyl capnines, 1-deoxy-1-sulfonic acid analogs of ceramides. The fatty acid methyl esters obtained from the lipids were heterogeneous, but in all cases were rich in hydroxylated fatty acyl groups, which constituted 66 to 95% of the total.

Many simple gliding bacteria contain significant quantities of phosphate-free, sulfur-containing lipids (sulfonolipids; N-acylamino-3-hydroxyisoheptadecane-1-sulfonic acids, or N-acyl capnines) that recently were shown to function in the ability of Cytophaga johnsonae to migrate over solid surfaces. Reported here is the synthesis, by surface-grown Cytophaga johnsonae cells, of two additional sulfonolipids not present in cells grown in liquid media. These newly characterized sulfonolipids are more polar than the N-acylcapnines characteristic of liquid grown cells. Acid methanolysis of the sulfonolipids revealed that the aminosulfonate capnine was common to all, thus indicating that the chemical differences in the compounds resided in their N-fatty acyl groups, and not in the aminosulfonate moiety. Instead of the non-hydroxy and 3-hydroxy fatty acyl moieties present in sulfonolipids of liquid-grown cells, one new sulfonolipid contained a 2-hydroxy, branched C15 fatty acid, while the other contained a 2,3-dihydroxy, isobranched C16 fatty acid, as indicated by gas chromatographic and mass spectrometric analyses. Although the structure of sulfonolipids thus varies between surface- and liquid-grown cells, no difference was found between the total quantity of sulfonolipids present under either of these conditions. The surface-dependent synthesis of these more polar N-acyl-aminosulfonates ceased immediately when surface-grown populations were suspended in broth. The ability of Cytophaga johnsonae to synthesize these compounds in response to a solid surface may be significant in relation to the organism's ability to migrate over such surfaces; it is one of few instances where a physical interaction of the cell surface has been shown to influence the molecular composition of a prokaryote.

We previously observed (W. Godchaux, L. Gorski, and E.R. Leadbetter, J. Bacteriol. 172:1250-1255, 1990) that two mutants (strains 21 and NS-1) of the gliding bacterium Cytophaga johnsonae that were totally deficient in motility-dependent colony spreading, movement of rafts (groups) of cells as observed with a microscope, and movement of polystyrene-latex spheres that attached to the cell surface (observed in wet mounts) were also deficient in a high-molecular-weight cell surface polysaccharide (HMPS) and suggested a role for that substance in gliding motility. Antisera have been prepared against the purified HMPS, and these were used to select mutants specifically and highly deficient in the polysaccharide. All five such mutants had rates of colony spreading and raft movement that were much lower than those of the parent strain, but the rate of increase in colony diameter was higher than that found for strains NS-1 and 21 (which do not undergo raft movement at all). Unlike these latter two strains, the HMPS mutants retained the ability to move polystyrene-latex spheres over their surfaces. Hence, HMPS deficiency results in defective motility but not nonmotility, and the HMPS deficiency cannot fully explain the phenotype of mutants 21 and NS-1; in these strains, gliding must be affected by additional biochemical lesions. The HMPS may, nonetheless, be advantageous in that it supports greater gliding speeds.

The lack of cell translocation and the resulting formation of nonspreading colonies of mutants of the gram-negative gliding bacterium Cytophaga johnsonae have been correlated with the loss of cell surface features of the organism. These cell surface traits include the ability to move polystyrene-latex beads over the cell surface and the ability to be infected by bacteriophages that infect the parent strain. In order to assess whether these traits reflect structures or functions that actually play a role in gliding, we studied a mutant (21A2I) selected for its inability to form spreading colonies; it is deficient in sulfonolipid, lacks bead movement ability, and is resistant to at least one bacteriophage. The provision of cysteate (a specific sulfonolipid precursor) restores lipid content and gliding to the mutant; hence, the lipids are necessary for motility. Growth with cysteate also restores bead movement and phage sensitivity. In order to determine the temporal relationship of these traits, we undertook a kinetic study of the appearance of them after addition of cysteate to the mutant. One predicts that appearance of a trait essential for cell translocation will either precede or accompany the appearance of this ability, while a nonessential trait need not do so. Sulfonolipid synthesis was the only trait that appeared before gliding; this is consistent with its established importance for motility. Bead movement and phage sensitivity first appeared only after gliding started, suggesting that the machinery involved in those processes is not necessary, at least for the initiation of gliding.

Phenol-extractable polysaccharides firmly associated with the outer membrane of the gliding bacterium Cytophaga johnsonae could be resolved by gel filtration in sodium dodecyl sulfate (SDS) or by SDS-polyacrylamide gel electrophoresis into a high-molecular-weight (H) fraction (excluded by Sephadex G-200) and a low-molecular-weight (L) fraction. Fraction L was rich in components typical of lipid A and the core region of lipopolysaccharide (P, 3-hydroxy fatty acids, and 2-keto-3-deoxyoctonate) and evidently was a lipopolysaccharide with a limited number of distal, repeating polysaccharide units, as judged by SDS-polyacrylamide gel electrophoresis. In relation to total carbohydrate, the H fraction was rich in amino sugar but poor in (possibly devoid of) the lipid A and core components. Two nongliding mutants were highly deficient in the H fraction; one of these was deficient in sulfonolipid but could be cured by provision of a specific sulfonolipid precursor, a process that also resulted in the return of both the H fraction and gliding, as well as the ability to move polystyrene latex spheres over the cell surface. Hence, the polysaccharide may be the component that is directly involved in motility, and the presence of sulfonolipids in the outer membrane is necessary for the synthesis or accumulation of the polysaccharide. This conclusion was reinforced by the fact that the second nongliding, polysaccharide-deficient mutant had a normal sulfonolipid content.

The gram-negative gliding bacterium Cytophaga johnsonae contains not only large quantities of unusual sulfonolipids but also, as we report here, a second class of unusual lipids. These lipids were detected and quantified by two-dimensional thin-layer chromatography of lipids from cells grown in the presence of [14C]acetate and shown by chemical studies to be alpha-N-(3-fatty acyloxy fatty acyl)ornithines. Like the sulfonolipids, these ornithine lipids were localized in the outer membrane (whereas phosphatidylethanolamine was the predominant lipid of the inner membrane). In a sulfonolipid-deficient mutant, the missing lipid was replaced, specifically, by an increased amount of ornithine lipid. Cells grown in liquid media contained predominantly ornithine lipids with nonhydroxylated residues in the O-fatty acyl position. In contrast, surface-grown cells contained a high proportion of ornithine lipids in which the O-fatty acyl group was 3-hydroxylated. The sulfonolipids and ornithine lipids are apparently coregulated in the sense that, regardless of perturbations caused by mutation or growth conditions, their total amounts remain constant at 40% of total cell lipid.

The characteristics of gliding bacteria isolated from both healthy and diseased sites in the oral cavity are, summarized and the taxonomic position of the bacteria discussed. Uniform attributes of the fusiform isolates include gliding motility, strictly fermentative metabolism dependent on the presence of CO2 (or HCO3-), under either anaerobic or aerobic conditions, presence of benzidine-reactive components, and the production of acetic and succinic acids as the major or sole, acidic, metabolic and products. Given the guanine and cytosine content of DNA, their gliding motility, and the ability of many strains to attack polysaccharide a relationship to the cytophagas is suggested. This relationship, along with the CO2-dependent growth is recognized by the generic name Capnocytophaga given them. Many of the isolates are grouped into three species C. ochracea, C. Sputigena, and C. gingivalis, separated on the basis of morphological and physiological traits.

Sixty-eight strains of capnophilic fusiform Gram-negative rods from the human oral cavity were subjected to extensive physiologic characterization, tested for susceptibility to various antibiotics, and the mol-percent guanine plus cytosine of each isolate determined. The characteristics of the isolates were compared with 10 fresh and 2 stock isolates of Fusobacterium nucleatum. The isolates clearly differed from the Fusobacterium species on the basis of molpercent guanine plus cytosine, end products, growth in a capnophilic environment and fermentation of carbohydrates.

Gram-negative, anaerobic gliding bacteria were isolated from normal supragingival plaque and from periodontal lesions. Isolates could be divided into two size classes: small 2.4–4.2 µm×0.38–0.5 µm and large 4.8–5.8 µm×0.42–0.6 µm cells. The outer membrane was either loose-fitting and wavy, or taut, and of variable thickness. An electron-dense fuzz was discernible on several of the isolates. The periplasmic region was of variable electron-density. The genus Capnocytophaga has been proposed for these organisms based on morphological and cultural characteristics.

Abstract: The enrichment and isolation in pure culture of a bacterium, identified as a strain of Desulfovibrio, able to release and reduce the sulfur of isethionate (2-hydroxyethanesulfonate) and other sulfonates to support anaerobic respiratory growth, is described. The sulfonate moiety was the source of sulfur that served as the terminal electron acceptor, while the carbon skeleton of isethionate functioned as an accessory electron donor for the reduction of sulfite. Cysteate (alanine-3-sulfonate) and sulfoacetaldehyde (acetaldehyde-2-sulfonate) could also be used for anaerobic respiration, but many other sulfonates could not. A survey of known sulfate-reducing bacteria revealed that some, but not all, strains tested could utilize the sulfur of some sulfonates as terminal electron acceptor. Isethionate-grown cells of Desulfovibrio strain IC1 reduced sulfonate-sulfur in preference to that of sulfate; however, sulfate-grown cells reduced sulfate-sulfur in preference to that of sulfonate.

Kinetic analyses of 14C-methylamine and 14C-bicarbonate assimilation in a gram-negative diplococcus suggests that methylamine assimilation occurs via a glycine-serine hydroxymethylation sequence rather than autotrophically by a Calvin cycle.

Electron microscopic examination of thin sections of vegetative cells and microcysts of S. myxococcoides indicates that the vegetative cells have a fine structure basically identical to that of other gram-negative bacteria. Microcysts, on the other hand, possess not only an extensive internal membrane system, an additional “intermediate layer” interposed between the plasma membrane and cell wall, but also a thick fibrillar outer coat or capsule.

The isolation of an ultraviolet absorbing material from spores of Sarcina ureae and its identification as dipicolinic acid (DPA) is described. The DPA was present in amounts ranging from 4–7% of the spore dry weight, but was not detected in vegetative cells.

1. The assimilatory nitrite reductase of the N2-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction of nitrite to NH3 with NADH as the electron donor. 3. NADH–nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect the enzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the microorganism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH3 is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase.

THE influence of nutrition on growth and (particularly) on morphogenesis of some fruiting myxobacteria was observed in this laboratory some two years ago. As recently reported1, a number of Myxococcus strains grow luxuriantly on an agar (2 per cent w/v) medium containing enzymatically hydrolysed casein (Difco ‘Tryptone’, 1 per cent w/v). However, only vegetative cells are present ; neither the formation of microcysts (cellular morphogenesis) nor of fruiting structures (colonial morphogenesis) takes place. Microcyst and fruiting body formation does occur, however, when the ‘Tryptone’ concentration is reduced to 0.1 or 0.2 per cent (w/v).

THE methane-utilizing bacterium Pseudomonas methanica oxidizes ethane to ethanol, acetaldehyde and acetic acid; propane to n-propanol, propionic acid and acetone; and n- butane to n-butanol, n-butyric acid and 2-butanone1. The formation of homologous oxygenated products made possible a study of the mechanism of the bacterial oxidation. Ethylene could not be detected as an intermediate during the conversion of ethane, nor was ethylene converted to the same products as ethane. Mass spectrometric analysis of the acetic acid and the acetaldehyde produced from deuteroethane (ethane-D6) eliminated the possibility that ethane was dehydrogenated to ethylene at a stage in the oxidation1.

Representative strains of two subspecies of Bacteroides melaninogenicus (subspecies melaninogenicus and subspecies intermedins) and Bacteroides asaccharolyticus as well as B. asaccharolyticus strain 536B isolated from a human perirectal abscess and Bacteroides fragilis ATCC 25285 were examined by glutaraldehyde-osmium fixation, ruthenium red fixation and staining, and thorium hydroxide staining as well as by the physical preparative techniques of critical point drying-transmission electron microscopy (CPD-TEM) and scanning electron microscopy (SEM). All strains, with the exception of B. fragilis 25285, possessed an electron-dense material external to their outer membranes. Ruthenium red staining further revealed a layer, external to the surface of the outer membrane, that was distinct for each species examined. Thorium hydroxide, as well as CPD-TEM and SEM, showed the cells to be interconnected by thin fibers that not only connected adjacent cells but also traversed several microns to connect cell aggregates.