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Biodegradation of ethylene glycol by a salt-requiring bacterium.

A gram-negative nonmotile rod which was capable of using 1,2-(14)C-ethylene glycol as a sole carbon source for growth was isolated from a brine pond, Great Salt Lake, Utah. The bacterium (ATCC 27042) required at least 0.85% NaCl for growth and, although the chloride ion was replaceable by sulfate ion, the sodium ion was not replaceable by potassium ion. The maximal concentration of salt tolerated for growth was approximately 12%. The bacterium was oxidase-negative when N,N-dimethyl-p-phenylenediamine was used and weakly positive when N,N,N',N'-tetramethyl-p-phenylenediamine was used. It grows on many sugars but does not ferment them, it does not have an exogenous vitamin requirement, and it possesses a guanine plus cytosine ratio of 64.3%. Incorporation of ethylene glycol carbon into cell and respired CO(2) was quantitated by use of radioactive ethylene glycol and a force-aerated fermentor. Glucose suppressed ethylene glycol metabolism. Cells grown on ethylene and propylene glycol respired ethylene glycol in a Warburg respirometer more rapidly than cells grown on glucose. Spectrophotometric evidence was obtained for oxidation of glycolate to glyoxylate by a dialyzed cell extract.

Alcohol Oxidoreductases↗

The probiotic bacterium Lactobacillus casei induces activation of the gut mucosal immune system through innate immunity.

The mechanisms by which probiotic bacteria affect the immune system are unknown yet, but many of them are attributed to an increase in the innate or in the acquired immune response. To study the influence of the probiotic bacterium Lactobacillus casei in the expression of receptors involved in the innate immune response, this bacterium was orally administered to BALB/c mice. After, they were sacrificed; the small intestine and intestinal fluids were collected to measure secretory immunoglobulin A (IgA) specific for L. casei. Mononuclear cells from Peyer's patches were isolated to determine the CD-206 and TLR-2 receptors. In histological slices we determined the number of IgA+, CD4+, CD8+, and CD3+ cells and two cytokines (interleulin-5 [IL-5] and IL-6). CD-206 and TLR-2 increased with respect to the untreated control. We did not observe an increase in the T population or in the IL-5-positive cells. IgA+ cells and IL-6-producing cells increased after 7 days of L. casei administration. We did not find specific antibodies against L. casei. The main immune cells activated after oral L. casei administration were those of the innate immune response, with an increase in the specific markers of these cells (CD-206 and TLR-2), with no modification in the number of T cells.

Administration, Oral↗

Bovine veneral vibriosis: antigenic variation of the bacterium during infection.

Host parasite relationships in the female genital tract were studied in bovine venereal vibriosis by investigating agglutinin production and alterations in superficial antigens of the bacterium during the course of infection in two heifers. Cervicovaginal mucus (CVM) steamed cell agglutinins were shown to appear earlier and remain at consistently higher levels than whole-cell agglutinins. Whole-cell agglutinin titers fluctuated much more than steamed cell titers, suggesting possible changes in whole-cell antigens. Marked antigenic variation was demonstrated in successive monthly CVM isolates from the two heifers by agglutination tests with rabbit antisera of various specificities. Some changes in CVM antibody specificity during the infection were noted also. Antigenic variation in the bacterium was proposed as a mechanism for maintenance of the asymptomatic cervicovaginal carrier state in the presence of antibody.

Agglutination Tests↗

Enhancement of experimental bacteremia and endocarditis caused by dysgonic fermenter (DF-2) bacterium after treatment with methylprednisolone and after splenectomy.

The dysgonic fermenter-2 bacterium is a newly recognized fastidious gram-negative bacillus that causes bacteremia and sometimes endocarditis in immunocompromised persons after they are bitten by dogs. To develop an experimental model of this infection, we placed polyethylene catheters across the aortic valves of New Zealand white rabbits, which were inoculated intravenously the next day with dysgonic fermenter-2 bacteria. After 1 week, the rabbits were killed and the endocardial vegetations were homogenized for quantitative culture. Large inocula (1.3 X 10(10) to 2.1 X 10(10) viable bacteria) were required to produce infected vegetations. All infected rabbits had negative blood cultures at the time of autopsy and most developed serum agglutinins against dysgonic fermenter-2 bacteria. Three daily injections of methylprednisolone (30 mg/kg), starting the day before inoculation, significantly increased the incidence of endocarditis and the number of bacteria per gram of infected vegetation (P less than 0.05). Treatment with methylprednisolone prolonged the initial bacteremia and caused significant increases in the numbers of bacteria per gram of blood, spleen, and liver compared with those of untreated controls (P less than 0.05). Rabbits that had previously undergone splenectomy showed prolongation of the initial bacteremia but no significant increase in the incidence of infected vegetations. These results showed that the dysgonic fermenter-2 bacterium is a pathogen that causes endocarditis in rabbits but that it requires a large inoculum and produces blood culture-negative infections. Treatment with methylprednisolone enhances infection by prolonging the initial bacteremia and probably by diminishing bactericidal activity in the vegetations.

Animals↗

Isolation of an intracellular bacterium from hamsters (Mesocricetus auratus) with proliferative ileitis and reproduction of the disease with a pure culture.

An intracellular bacterium was isolated from hamsters (Mesocricetus auratus) with proliferative ileitis. The organism was isolated in Intestine 407 and GPC-16 cell cultures (incubated in a microaerophilic atmosphere) from isolated and lysed epithelial cells from hamsters with proliferative ileitis. The bacterium measured 1.4 to 1.7 microns in length by 0.26 to 0.34 microns in width, was slightly curved, and had an irregular trilaminar cell wall. Inoculation of hamsters with a cell culture lysate containing the organism or a 0.65-microns-pore-size filtrate of an infected-cell lysate resulted in the typical lesions of proliferative ileitis in approximately 50% of the animals in 28 days. Hamsters inoculated with uninfected cells or a 0.2-microns-pore-size filtrate of an infected-cell lysate remained uninfected. Attempts to propagate the organism on cell-free media have been unsuccessful.

Animals↗

Neutrophils are critical for host defense against primary infection with the facultative intracellular bacterium Francisella tularensis in mice and participate in defense against reinfection.

It is generally believed that immunity to experimental infection with the facultative intracellular bacterium Francisella tularensis is an example of T-cell-mediated immunity that is expressed by activated macrophages and mediated by Francisella-specific T cells. According to the results presented herein, neutrophils are also essential for defense against primary infection with this organism. It is shown that mice depleted of neutrophils by treatment with the granulocyte-specific monoclonal antibody RB6-8C5 are rendered defenseless against otherwise sublethal doses of F. tularensis LVS inoculated intravenously or intradermally. In neutrophil-depleted mice, the organism grew progressively in the livers, spleens, and lungs to reach lethal numbers, whereas infection was resolved in normal mice. Although neutrophils were found to resistance to reinfection, their participation was less important. The results suggest that neutrophils are needed for defense against primary infection because they serve to restrict the growth of F. tularensis before it reaches numbers capable of overwhelming a developing specific immune response. The exact way that neutrophils achieve this is not clear at this time, although it is probable that they contribute in ways other than by ingesting and killing the bacterium.

Animals↗

Induction of apoptotic cell death in peripheral blood mononuclear and polymorphonuclear cells by an oral bacterium, Fusobacterium nucleatum.

It is largely unknown why a variety of bacteria present in the oral cavity are capable of establishing themselves in the periodontal pockets of nonimmunocompromised individuals in the presence of competent immune effector cells. In this paper we present evidence for the immunosuppressive role of Fusobacterium nucleatum, a gram-negative oral bacterium which plays an important role in the generation of periodontal disease. Our studies indicate that the immunosuppressive role of F. nucleatum is largely due to the ability of this organism to induce apoptotic cell death in peripheral blood mononuclear cells (PBMCs) and in polymorphonuclear cells (PMNs). F. nucleatum treatment induced apoptosis of PBMCs and PMNs as assessed by an increase in subdiploid DNA content determined by DNA fragmentation and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling assays. The ability of F. nucleatum to induce apoptosis was abolished by either heat treatment or proteinase digestion but was retained after formaldehyde treatment, suggesting that a heat-labile surface protein component is responsible for bacterium-mediated cell apoptosis. The data also indicated that F. nucleatum-induced cell apoptosis requires activation of caspases and is protected by NF-kappaB. Possible mechanisms of F. nucleatum's role in the pathogenesis of periodontal disease are discussed.

Apoptosis↗

In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 subunit of interleukin-12 (IL-12) but not on IL-12 p70.

To determine the role of interleukin-12 (IL-12) in primary and secondary immunity to a model intracellular bacterium, we have comprehensively evaluated infection with Francisella tularensis LVS in three murine models of IL-12 deficiency. Mice lacking the p40 protein of IL-12 (p40 knockout [KO] mice) and mice treated in vivo with neutralizing anti-IL-12 antibodies survived large doses of primary and secondary LVS infection but never cleared bacteria and exhibited a chronic infection. In dramatic contrast, mice lacking the p35 protein (p35 KO mice) of heterodimeric IL-12 readily survived large doses of primary sublethal LVS infection as well as maximal secondary lethal challenge, with only a slight delay in clearance of bacteria. LVS-immune wild-type (WT) lymphocytes produced large amounts of gamma interferon (IFN-gamma), but p35 KO and p40 KO lymphocytes produced much less; nonetheless, similar amounts of NO were found in all cultures containing immune lymphocytes, and all immune lymphocytes were equally capable of controlling intracellular growth of LVS in vitro. Purified CD4(+) and CD8(+) T cells from both WT and p40 KO mice controlled intracellular growth, even though T cells from WT mice produced much more IFN-gamma than those from p40 KO mice, and p40 KO T cells did not adopt a Th2 phenotype. Thus, while IL-12 p70 stimulation of IFN-gamma production may be important for bacteriostasis, IL-12 p70 is not necessary for appropriate development of LVS-immune T cells that are capable of controlling intracellular bacterial growth and for clearance of primary or secondary LVS infection. Instead, an additional mechanism dependent on the IL-12 p40 protein, either alone or in another complex such as the newly discovered heterodimer IL-23, appears to be responsible for actual clearance of this intracellular bacterium.

Animals↗

Moraxella catarrhalis bacterium without endotoxin, a potential vaccine candidate.

Lipooligosaccharide (LOS) is a major surface component of Moraxella catarrhalis and a possible virulence factor in the pathogenesis of human infections caused by this organism. The presence of LOS on the bacterium is an obstacle to the development of vaccines derived from whole cells or outer membrane components of the bacterium. An lpxA gene encoding UDP-N-acetylglucosamine acyltransferase responsible for the first step of lipid A biosynthesis was identified by the construction and characterization of an isogenic M. catarrhalis lpxA mutant in strain O35E. The resulting mutant was viable despite the complete loss of LOS. The mutant strain showed significantly decreased toxicity by the Limulus amebocyte lysate assay, reduced resistance to normal human serum, reduced adherence to human epithelial cells, and enhanced clearance in lungs and nasopharynx in a mouse aerosol challenge model. Importantly, the mutant elicited high levels of antibodies with bactericidal activity and provided protection against a challenge with the wild-type strain. These data suggest that the null LOS mutant is attenuated and may be a potential vaccine candidate against M. catarrhalis.

Acyltransferases↗

Whole-genome transcription profiling reveals genes up-regulated by growth on fucose in the human gut bacterium "Roseburia inulinivorans".

"Roseburia inulinivorans" is an anaerobic polysaccharide-utilizing firmicute bacterium from the human colon that was identified as a producer of butyric acid during growth on glucose, starch, or inulin. R. inulinivorans A2-194 is also able to grow on the host-derived sugar fucose, following a lag period, producing propionate and propanol as additional fermentation products. A shotgun genomic microarray was constructed and used to investigate the switch in gene expression that is involved in changing from glucose to fucose utilization. This revealed a set of genes coding for fucose utilization, propanediol utilization, and the formation of propionate and propanol that are up-regulated during growth on fucose. These include homologues of genes that are implicated in polyhedral body formation in Salmonella enterica. Dehydration of the intermediate 1,2-propanediol involves an enzyme belonging to the new B12-independent glycerol dehydratase family, in contrast to S. enterica, which relies on a B12-dependent enzyme. A typical gram-positive agr-type quorum-sensing system was also up-regulated in R. inulinivorans during growth on fucose. Despite the lack of genome sequence information for this commensal bacterium, microarray analysis has provided a powerful tool for obtaining new information on its metabolic capabilities.

Bacteria, Anaerobic↗

Demonstration by freeze-etching of a single cleavage plane in the cell wall of a gram-negative bacterium.

In the examination of protoplasts of a gram-negative bacterium classified as a Pseudomonas sp. by freeze-etching, we found a smooth external surface which is not seen if the preparations are not "etched." This external structure is seen as a sleeve surrounding and connecting the cells in unetched preparations, and we present evidence that it is a eutectic formed during the freezing of the specimen. In the system used in this study, the four layers of the cell wall of a gram-negative bacterium can be removed from the cell. The single cell wall cleavage plane is not affected by the removal of the loosely bound outer layer or of the peptidoglycan layer, but it is lost when the outer double track layer and the underlying soluble layer are simultaneously removed. Thus, we conclude that it is one of these two layers which is responsible for the cleavage plane which exposes variable areas of a smooth surface in the cell wall. This cell wall cleavage plane is more likely to deflect the actual cleavage of the frozen cell when cells are relatively old or when they are suspended in sucrose.

Bacteriological Techniques↗

Whole-cell and membrane lipids of the methylotrophic bacterium Methylosinus trichosporium.

The lipid composition of the methylotrophic bacterium Methylosinus trichosporium was examined. Whole-cell lipid distribution was 39.1% neutral lipids, 34.5% polar lipids, and 26.4% poly-beta-hydroxybutyric acid. Membrane lipids were 83% phospholipids, with phosphatidylethanolamine and phosphatidylglycerol accounting for over 94% of the total. All the phospholipids had similar fatty acid compositions, with 18:1 accounting for about 87% of the total and most of the rest consisting of 16:1. Similarities between the lipid composition of this bacterium and other bacteria are discussed.

Cell Membrane↗

Novel bacterium infecting an African snail.

Nodules found in the superficial tissues of laboratory-maintained snails (Bulinus jousseaumei) contained a bacterium of two forms. This nonmotile microorganism occurred in intracellular packets as a simple gram-negative rod that appeared to undergo intrapacket transition to a cephalotrichous form. The latter is characterized by a "head" from which emerge long, thick, rigid, flagella-like, helically constituted filamentous organelles with a core and an outer component that is not an extension of the bacterial envelope. Neither form was successfully cultured, but clean snails derived from eggs removed before hatching developed nodules within 1 to 3 months of exposure to infected snails. The infectivity was specific for the host snail, and no transmission occurred to snails of 5 other genera tested. The presence of nodules did not interfere with longevity or reproduction of infected snails. Details of infectivity, transition, and taxonomic position of the bacterium remain to be explored, but it is reported because of unique morphological and ultrastructural features not previously known.

Africa↗

Transfer of nitrogen fixation genes from a bacterium with the characteristics of both Rhizobium and Agrobacterium.

Strain T1K, reported to be Rhizobium trifolii strain T1 carrying the drug resistance plasmid RU-1drd, was able to transfer a cluster of nif+ genes to Escherichia coli K-12. Additional genetic material, resembling the gal-chlA region of E. coli, was also transferred from strain T1K. The segregation pattern of these transferred genes suggested that they were on a plasmid. Although strain TIK was able to nodulate red and white clover, it also formed very slow-growing galls on tomato stems and shared many physiological properties with Agrobacterium tumefaciens, to which it seemed more closely related than to R. trifolii. The R. trifolii hybrid T1 (R1-19drd), constructed by conjugation, did not share any of these properties of both A. tumefaciens. Thus, strain T1K appears to be a bacterium with properties of both A. tumefaciens and R. trifolii and with the capacity to transfer nif+ genes and other functions which it may have "cloned" from another bacterium such as Klebsiella.

Chromosomes, Bacterial↗

Growth of the photosynthetic bacterium Rhodopseudomonas capsulata chemoautotrophically in darkness with H2 as the energy source.

The phototrophic bacterium Rhodopseudomonas capsulata was found to be capable of growing chemoautotrophically under aerobic conditions in darkness. Growth was strictly dependent on the presence of H2 as the source of energy and reducing power, O2 as the terminal electron acceptor for energy transduction, and CO2 as the sole carbon source; under optimal conditions the generation time was about 6 h. Chemoautotrophically grown cells showed a relatively high content of bacteriochlorophyll a and intracytoplasmic membranes (chromatophores). Experiments with various mutants of R. capsulata, affected in electron transport, indicate that either of the two terminal oxidases of this bacterium can participate in the energy-yielding oxidation of H2. The ability of R. capsulata to multiply in at least five different physiological growth modes suggests that it is one of the most metabolically versatile procaryotes known.

Bacterial Chromatophores↗

Fixation of dinitrogen derived from denitrification of nitrate in a photosynthetic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans.

Studies with 15N demonstrated that the phototrophic bacterium Rhodopseudomonas sphaeroides forma sp. denitrificans strain IL106 cannot assimilate NO-3 but rather denitrifies it to N2. This strain also fixed N2 into cell protein, although nitrogenase activity was partially inhibited in the presence of NO-3. Strain IL106 did not assimilate NO-3, but growing cultures and washed cell suspensions incorporated the tracer from 15NO-3 via denitrification to 15N2 and then via nitrogenase into cell nitrogen. This incorporation was inhibited in cells supplied with NH4+ or in the absence of light, thus confirming the participation of nitrogenase in the assimilation of nitrogen from nitrate. This represents a novel type of N2 recycling in a photodiazotrophic denitrifying bacterium.

Bacterial Proteins↗

Proton efflux coupled to dark H2 oxidation in whole cells of a marine sulfur photosynthetic bacterium (Chromatium sp. strain Miami PBS1071).

Whole cells of photoanaerobically grown Chromatium sp. strain Miami PBS1071, a marine sulfur purple bacterium, oxidized H2 in the dark through the oxyhydrogen reaction at rates of up to 59 nmol of H2 per mg (dry weight) per min. H2 oxidation was routinely measured in H2 pulse experiments with air-equilibrated cells. The reaction was accompanied by a reversible H+ efflux from the cells, suggesting an outward H+ translocation reaction coupled to H2 oxidation. The H+/e- ratio, calculated from simultaneous measurements of H2, O2, and H+ changes in the medium, varied with the cultures from 0.7 to 1.2. The ratio increased considerably when the backflow of H+ was taken into account. Anaerobic H2 uptake with 2,5-dimethyl-p-benzoguinone as an oxidant also showed a weak H+-translocating activity. No H+-translocating activity was detected with methylene blue as an oxidant. Carbonylcyanide 3-chlorophenylhydrazone (1 microM) stimulated H2 oxidation and abolished the associated H+ changes when H2 oxidation was observed in O2 pulse experiments with H2-Ar-equilibrated cells. However, the uncoupler inhibited both H2 oxidation and H+ changes when measurements were made in H2 pulse experiments with air-equilibrated cells. It is suggested that in this bacterium the susceptibility of hydrogenase to reversible O2 inactivation in situ is enhanced by the presence of uncoupling agents.

Benzoquinones↗

Development of a gene cloning system for the hydrogen-producing marine photosynthetic bacterium Rhodopseudomonas sp.

Seventy-six strains of marine photosynthetic bacteria were analyzed by agarose gel electrophoresis for plasmid DNA content. Among these strains, 12 carried two to four different plasmids with sizes ranging from 3.1 to 11.0 megadaltons. The marine photosynthetic bacterium Rhodopseudomonas sp. NKPB002106 had two plasmids, pRD06S and pRD06L. The smaller plasmid, pRD06S, had a molecular weight of 3.8 megadaltons and was cut at a single site by restriction endonucleases SalI, SmaI, PstI, XhoI, and BglII. Moreover, the marine photosynthetic bacterium Rhodopseudomonas sp. NKPB002106 containing plasmid pRD06 had a satisfactory growth rate (doubling time, 7.5 h), a hydrogen-producing rate of 0.96 mumol/mg (dry weight) of cells per h, and nitrogen fixation capability. Plasmid pRD06S, however, had neither drug resistance nor heavy-metal resistance, and its copy number was less than 10. Therefore, a recombinant plasmid consisting of pRD06S and Escherichia coli cloning vector pUC13 was constructed and cloned in E. coli. The recombinant plasmid was transformed into Rhodopseudomonas sp. NKPB002106. As a result, Rhodopseudomonas sp. NKPB002106 developed ampicillin resistance. Thus, a shuttle vector for gene transfer was constructed for marine photosynthetic bacteria.

Ampicillin↗