Ribotyping to differentiate Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme isolated from bovine ruminal contents and liver abscesses.

Differences in biological activities (hemagglutination, hemolytic, leukotoxic, and virulence) and ribotypes between the two subspecies of Fusobacterium necrophorum of bovine ruminal and liver abscess origins were investigated. Hemagglutination activity was present in all hepatic, but only some ruminal, strains of Fusobacterium necrophorum subsp. necrophorum. Ruminal F. necrophorum subsp. necrophorum had low leukotoxin titers yet was virulent in mice. Fusobacterium necrophorum subsp. funduliforme of hepatic or ruminal origin had no hemagglutination activity, had low hemolytic and leukotoxic activities, and was less virulent to mice. For ribotyping, chromosomal DNAs of 10 F. necrophorum subsp. necrophorum and 11 F. necrophorum subsp. funduliforme isolates were digested with restriction endonucleases (EcoRI, EcoRV, SalI, PstI, and HaeIII) and examined by restriction fragment length polymorphisms after hybridizing with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzyme for ribotyping was EcoRI. The presence or absence of two distinct bands of 2.6 and 4.3 kb differentiated the two subspecies. Regardless of the origin, only F. necrophorum subsp. necrophorum, a virulent subspecies, had a ca. 2.6-kb band, whereas F. necrophorum subsp. funduliforme, a less virulent subspecies, had a ca. 4.3-kb band. Ribotyping appears to be a useful technique to genetically differentiate the two subspecies of F. necrophorum.     

Chemical composition of endotoxins produced by Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme

The endotoxins from two recently-classified subspecies of Fusobacterium, namely F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, were compared. Chemical analysis of the isolated endotoxins revealed that they were clearly different. Distinct levels of polysaccharides were demonstrated. The endotoxins isolated were devoid of heptose and 3-deoxy-D-manno-octulosonate (KDO). The endotoxins of F. n. necrophorum and F. n. funduliforme contained lipid A in a ratio of 4:1 which may account for the variations in their virulence.     

Site specific endonuclease from Fusobacterium nucleatum.

Four different isolates of Fusobacterium nucleatum (A,C,D and E) contain restriction endonucleases of differing specificity. Whilst many of the endonucleases are isochizomers of known enzymes, two novel activities are Fnu DII which recognizes and cleaves the sequence 5'-CGCT-3'/3'-GCGC-5' AND Fnu EI which recognizes and cleaves the sequence 5'-GATC-3'/3'-CTAG-5' irrespective of the extent of methylation of the adenine residues.     

Ribotyping of Fusobacterium necrophorum strains isolated from bovine and ovine hepatic abscesses

A microbiological study was made of 100 strains of Fusobacterium necrophorum isolated from hepatic abscesses in bovine and ovine herds. Differences between the biological activity and ribotypes within the two F. necrophorum subspecies were studied. Conventional methods identified 89 isolates as F. necrophorum subsp. necrophorum and 11 as F. necrophorum subsp. funduliforme. For ribotyping, 50 strains (35 F.n. subsp. necrophorum, 11 F.n. subsp. funduliforme and 4 reference strains) were digested with restriction endonucleases (HindIII, EcoRI and BamHI) and examined after hybridization with digoxigenin-labelled cDNA probe transcribed from a 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzymes for ribotyping were EcoRI and BamHI. The presence or absence of two distinct band of 5 kb (EcoRI) and 10.5 kb (BamHI) differentiated the two subspecies. This technique also revealed genetic differences between isolates which could be used in the epidemiological study of clinical processes caused by F. necrophorum.     

Extraction and properties of hemagglutinin from cell wall fragments of Fusobacterium nucleatum.

To study the hemagglutinin of Fusobacterium nucleatum, methods were sought to solubilize and purify this component. When cells of F. nucleatum were ruptured by passage through a French press, the fragments lost virtually all ability to agglutinate human erythrocytes. Extraction of the fragments with 2% Triton X-100 for 30 min at 22 degrees C restored hemagglutinating activity (HA). Hemagglutination by these fragments could be inhibited by arginine, as can hemagglutination by intact bacteria. Treatment of active cell wall fragments with pronase and 2% Triton X-100-EDTA at 37 degrees C or with pronase and 0.1% Triton X-100-EDTA at pH 10.0 allowed recovery of solubilized HA. The former HA was inhibited by arginine (arg+) whereas the latter was not (arg-). Fractionation of the arg+ extract by preparative isoelectric focusing showed that HA was recovered from the gel sections having a pH between 4.5 and 5.5. Hemagglutination by this preparation was still arg+. Chromatography of this hemagglutinin on DEAE-Sephadex increased the specific activity to high levels with a loss of inhibition by arginine. A fraction from the DEAE-Sephadex column containing 10,700 HA units per mg of protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Solubilization at 22 degrees C before electrophoresis revealed three Coomassie blue-staining bands which migrated with apparent molecular weights of about 21,000, 38,000 and 60,000. When the same DEAE fraction was boiled in sodium dodecyl sulfate, electrophoresis revealed only one band with an apparent molecular weight of 21,000.     

Fusobacterium nucleatum subsp. fusiforme subsp. nov. and Fusobacterium nucleatum subsp. animalis subsp. nov. as additional subspecies within Fusobacterium nucleatum.

Using a variety of physiological, biochemical, and molecular systematic analyses, we have shown previously that there are four groups within the species Fusobacterium nucleatum. Two of these groups of strains correspond to the recently proposed taxa F. nucleatum subsp. nucleatum and F. nucleatum subsp. polymorphum. In this paper we show that the two remaining groups are distinct and formally propose that they should be recognized as F. nucleatum subsp. fusiforme (type strain, NCTC 11326) and F. nucleatum subsp. animalis (type strain, NCTC 12276). The tests which we used did not allow a full assessment of the status of F. nucleatum subsp. vincentii compared with F. nucleatum subsp. nucleatum.     

Collagenolytic activity of a cell wall preparation from Fusobacterium necrophorum subsp. necrophorum

A collagenolytic preparation of Fusobacterium necrophorum subsp. necrophorum was derived from the bacterial cell. It was further treated for gel permeation with Toyopearl HW 50, followed by Sepharose 4B column chromatography. In sodium dodecyl sulphate polyacrylamide gel electrophoresis, the final preparation exhibited one definite band and at least one faint band. It was inactivated completely by adjusting the pH to 4.0 or by heating at 80 degrees C for 30 min.     

Peptidoglycan precursor from Fusobacterium nucleatum contains lanthionine.

Fusobacterium nucleatum was grown in the presence of [14C]UDP. By means of sequential precipitation and chromatographic separation of the cytoplasmic content, a peptidoglycan [14C]UDP pentapeptide containing lanthionine was isolated. This finding indicates that lanthionine is synthesized and incorporated as such during the assembly of the peptidoglycan.     

Adherence of Fusobacterium necrophorum subsp. necrophorum to ruminal cells derived from bovine rumenitis

Fusobacterium necrophorum subsp. necrophorum strain VPI 2891 was shown to adhere to the surfaces of ruminal cells derived from bovine rumenitis. The strain also attached to bovine type 1 collagen. Treatment of the bacterium with antiserum to bacterial cells reduced attachment. The bacterial attachment was also markedly reduced when the ruminal cells had been pretreated with anticollagen serum. Fluorescence specific for the collagen was demonstrated on the surface of bovine tissue affected with rumenitis. These findings suggest that F. necrophorum subsp. necrophorum strain VPI 2891 adheres to the ruminal cells derived from rumenitis tissue and that the attachment may be mediated by cellular collagen.     

Location of haemagglutinin in bacterial cells of Fusobacterium necrophorum subsp. necrophorum.

The location of haemagglutinin (HA) of Fusobacterium necrophorum subsp. necrophorum VPI 2891 strain was investigated by immunofluorescence, confocal laser scan microscopy and immunoelectron microscopy. The immunofluorescence study demonstrated the fluorescence specific for the HA on the bacterial cells and confocal laser scan microscopy indicated similar fluorescence around the cross section of the bacterial cell. The immunoelectron microscopic study also revealed that the protein A-gold conjugates were located around the bacterial surfaces. These findings suggest that HA is one of the components of the cell surfaces of F. necrophorum subsp, necrophorum.     

The biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase from Fusobacterium nucleatum (subsp. nucleatum)

Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na⁺ (K _m=3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent K _m for Na⁺ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, (m=65 kDa), (33 kDa), (19 kDa), and (16 kDa) with the functions of a carboxy transferase (), a carboxy lyase ( and probably ) and a biotin carrier (). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the - and the biotin containing subunits of the three decarboxylases and that from Peptostreptoccus asaccharolyticus could be detected on Western blots.     

Characterisation and subcellular localisation of the GroEL-like and DnaK-like proteins isolated from Fusobacterium nucleatum ATCC 10953

Fusobacterium nucleatum is associated with periodontitis in humans, and is a central member of the dental biofilm. Heat shock proteins (HSPs) of many different bacteria have been considered to play important roles during inflammations and infections. We have identified and characterised the HSP60 and HSP70, the Escherichia coli GroEL and DnaK homologues, respectively, in F. nucleatum ATCC 10953. The N-terminal 22 amino acid residues of HSP60 exhibited up to 63.6% identity with members of the HSP60 heat shock protein family of some selected bacterial species, while the N-terminal of 25 residues of HSP70 revealed up to 80% identity with members of the HSP70 family. The subcellular localisation of HSP60 and HSP70 was analysed by immunoblotting of bacterial cell fractions and immunoelectron microscopy of whole cells. HSP60 and HSP70 were localised in the cytosol, associated with membranes and extracellular fractions. These results are consistent with localisation for HSPs found in other micro-organisms, which further lead to the suggestion of a potential role in the pathogenesis of infectious diseases.     

Pathway of lysine degradation in Fusobacterium nucleatum.

Lysine was fermented by Fusobacterium nucleatum ATCC 25586 with the formation of about 1 mol each of acetate and butyrate. By the use of [1-14C]lysine or [6-14C]lysine, acetate and butyrate were shown to be derived from both ends of lysine, with acetate being formed preferentially from carbon atoms 1 and 2 and butyrate being formed preferentially from carbon atoms 3 to 6. This indicates that the lysine carbon chain is cleaved between both carbon atoms 2 and 3 and carbon atoms 4 and 5, with the former predominating [1-14C]acetate was also extensively incorporated into butyrate, preferentially into carbon atoms 3 and 4. Cell-free extracts of F. nucleatum were shown to catalyze the reactions of the 3-keto,5-aminohexanoate pathway of lysine degradation, previously described in lysine-fermenting clostridia. The 3-keto,5-aminohexanoate cleavage enzyme was partially purified and shown to have properties much like those of the clostridial enzyme. We conclude that both the pathway and the enzymes of lysine degradation are similar in F. nucleatum and lysine-fermenting clostridia.     

2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein

Anaerobically prepared cell-free extracts from Fusobacterium nucleatum contain 2-hydroxyglutaryl-CoA dehydratase with a specific activity of 20 nkat mg^-1. The enzyme was purified 24-fold to a specific activity of 480 nkat mg^-1 by anion exchange chromatography, gel filtration and chromatography on Blue-Sepharose. The activity of the purified enzyme was strictly dependent on the reductant Ti(III)citrate and stimulated 25-fold by 0.15 mM ATP and 5 mM MgCl₂. ATP is hydrolysed to ADP during incubation with 2-hydroxyglutaryl-CoA dehydratase in the presence or absence of the substrate. The enzyme is extremely sensitive towards oxygen and is inhibited by 10 M chloramphenicol, 10 M 2,4-dinitrophenol or 0.15 mM hydroxylamine. The pure enzyme consists of three subunits (49 kDa), (39 kDa) and (24 kDa) in approximately equal amounts. In this respect the enzyme differs from the related 2-hydroxy-glutaryl-CoA dehydratase from Acidaminococcus fermentans and lactyl-CoA dehydratase from Clostridium propionicum both of which are composed of only two subunits with sizes comparable to those of and but require an additional protein for activity. The relative molecular mass of the native enzyme of about 100 kDa suggests a trimeric -structure. The homogeneous enzyme contains riboflavin (0.5 mol/112 kDa), iron and sulfur (3.5 mol/112 kDa each). Polyclonal antibodies directed against the 2-hydroxyglutaryl-CoA dehydratase from A. fermentans did not crossreact with cell free extracts or purified dehydratase from F. nucleatum. A comparison of the N-terminal amino acid sequences of the dehydratase subunits from A. fermentans and F. nucleatum, however, showed some similarities in the -subunits.Non-standard abbreviations DTT dithiothreitol - PAGE polyaccrylamide gel electrophoresis - VIS visible     

Partial characterization of Fusobacterium necrophorum protease

Partial characterization of Fusobacterium necrophorum protease was investigated. The protease was partially purified by gel filtration with Toyopearl HW 55. The final preparation was inactivated completely by heating at 60 degrees C for 30 min and inhibited by ascorbic acid, sodium thioglycollate and p-hydromercuribenzoate. Antibody response to the protease was demonstrated in mice receiving 10(4) CFU of F. necrophorum.     

Fusobacterium nucleatum subsp. fusiforme Gharbia and Shah 1992 is a Later Synonym of Fusobacterium nucleatum subsp. vincentii Dzink et al. 1990

On the basis of the DNA–DNA hybridization patterns and phenotypic characteristics, Fusobacterium nucleatum was classified into five subspecies. Previous studies have suggested that F. nucleatum subsp. vincentii is genetically similar to F. nucleatum subsp. fusiforme. The aim of this study was to investigate the possibility of classifying these two subspecies into a single subspecies by phylogenetic analysis using a single sequence (24,715 bp) concatenated 22 housekeeping genes of eight F. nucleatum strains including type strains of five F. nucleatum subspecies. The phylogenetic analysis indicated that F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme were clustered in the same group and each strain of other F. nucleatum subspecies were also separated into the same cluster. These results suggested that F. nucleatum subsp. fusiforme and F. nucleatum subsp. vincentii can be classified into a single subspecies. F. nucleatum subsp. vincentii was early published name; therefore, F. nucleatum subsp. fusiforme Gharbia and Shah 1992 can be regarded as a later synonym of F. nucleatum subsp. vincentii Dzink et al. 1990.     

Haemagglutination and haemolysis by oral Fusobacterium nucleatum

Haemagglutination and haemolytic activity of 80 Fusobacterium nucleatum isolates from human and animal origin, on different human blood types was evaluated. All the isolates were able to agglutinate erythrocytes and the most were either alpha-haemolytic or beta-haemolytic. No specificity between haemolysin or haemagglutinin and blood type was observed. Haemagglutination activity was inhibited when D-galactose, D-lactose or D-raffinose were used. Haemagglutination and haemolysis may be important factors in the pathogenesis of human and animal periodontal diseases.     

Hydrophobicity of Fusobacterium necrophorum biovars A and B

Abstract Biovar A strains of Fusobacterium necrophorum exhibited high hydrophobicity when examined by the method of Rosenberg et al. Biovar B strains showed a lower cell surface hydrophobicity than biovar A strains. Biovar B strains were divided into 2 groups according to their hydrophobic activity. The strains of biovar A and the first group of biovar B were increasingly removed from aqueous phase to octane phase by increasing the volume of octane, but the turbidity of the second group of biovar B was not significantly affected. The hydrophobicity of biovar A strains decreased on heating at 60 and 100°C for 30 min.