Structure and function of Bs164 ß-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164.

Recent work exploring protein sequence space has revealed a new glycoside hydrolase (GH) family (GH164) of putative mannosidases. GH164 genes are present in several commensal bacteria, implicating these genes in the degradation of dietary glycans. However, little is known about the structure, mechanism of action, and substrate specificity of these enzymes. Herein we report the biochemical characterization and crystal structures of the founding member of this family (Bs164) from the human gut symbiont Bacteroides salyersiae. Previous reports of this enzyme indicated that it has α-mannosidase activity, however, we conclusively show that it cleaves only ß-mannose linkages. Using NMR spectroscopy, detailed enzyme kinetics of WT and mutant Bs164, and multiangle light scattering we found that it is a trimeric retaining ß-mannosidase, that is susceptible to several known mannosidase inhibitors. X-ray crystallography revealed the structure of Bs164, the first known structure of a GH164, at 1.91 Å resolution. Bs164 is composed of three domains: a (ß/α)8 barrel, a trimerization domain, and a ß-sandwich domain, representing a previously unobserved structural-fold for ß-mannosidases. Structures of Bs164 at 1.80-2.55 Å resolution in complex with the inhibitors noeuromycin, mannoimidazole, or 2,4-dinitrophenol 2-deoxy-2-fluoro-mannoside reveal the residues essential for specificity and catalysis including the catalytic nucleophile (Glu-297) and acid/base residue (Glu-160). These findings further our knowledge of the mechanisms commensal microbes use for nutrient acquisition.

Outer membrane vesicles blebbing contributes to B. vulgatus mpk-mediated immune response silencing.

The Gram negative intestinal symbiont Bacteroides vulgatus mpk is able to prevent from induction of colonic inflammation in Rag1-/- mice and promotes immune balance in Il2-/- mice. These inflammation-silencing effects are associated with B. vulgatus mpk-mediated induction of semi-mature dendritic cells, especially in the colonic lamina propria (cLP). However the beneficial interaction of bacteria with host immune cells is limited due to the existence of a large mucus layer covering the intestinal epithelium. How can intestinal bacteria overcome this physical barrier and contact the host immune system? One mechanism is the production of outer membrane vesicles (OMVs) via ubiquitous blebbing of the outer membrane. These proteoliposomes have the ability to traverse the mucus layer. Hence, OMVs play an important role in immunomodulation and the maintenance of a balanced gut microbiota. Here we demonstrate that the stimulation of bone marrow derived dendritic cells (BMDCs) with isolated OMVs originated from B. vulgatus mpk leads to the induction of a tolerant semi-mature phenotype. Thereby, microbe- associated molecular patterns (MAMPs) delivered by OMVs are crucial for the interaction and the resulting maturation of immune cells. Additional to the binding to host TLR4, a yet unknown ligand to TLR2 is indispensable for the conversion of immature BMDCs into a semi-mature state. Thus, crossing the epithelial mucus layer and directly contact host cells, OMV mediate cross-tolerance via the transport of various Toll-like receptor antigens. These features make OMVs to a key attribute of B. vulgatus mpk for a vigorous acellular prevention and treatment of systemic diseases.

Paneth cell α-defensin 6 (HD-6) is an antimicrobial peptide.

Defensins protect human barriers from commensal and pathogenic microorganisms. Human α-defensin 6 (HD-6) is produced exclusively by small intestinal Paneth cells but, in contrast to other antimicrobial peptides (AMPs) for HD-6, no direct antibacterial killing activity has been detected so far. Herein, we systematically tested how environmental factors, like pH and reducing conditions, affect antimicrobial activity of different defensins against anaerobic bacteria of the human intestinal microbiota. Remarkably, by mimicking the intestinal milieu we detected for the first time antibacterial activity of HD-6. Activity was observed against anaerobic gut commensals but not against some pathogenic strains. Antibiotic activity was attributable to the reduced peptide and independent of free cysteines or a conserved histidine residue. Furthermore, the oxidoreductase thioredoxin, which is also expressed in Paneth cells, is able to reduce a truncated physiological variant of HD-6. Ultrastructural analyses revealed that reduced HD-6 causes disintegration of cytoplasmic structures and alterations in the bacterial cell envelope, while maintaining extracellular net-like structures. We conclude that HD-6 is an antimicrobial peptide. Our data suggest two distinct antimicrobial mechanisms by one peptide: HD-6 kills specific microbes depending on the local environmental conditions, whereas known microbial trapping by extracellular net structures is independent of the reducing milieu.

Anti-stress proteins produced by Bacteroides thetaiotaomicron after nutrient starvation.

Bacteroides thetaiotaomicron maybe one of the most adaptable intestinal bacteria due to its complex genome. Known to be an opportunistic pathogenic anaerobe, B. thetaiotaomicron has recently been described as a symbiont with anti-inflammatory properties. In this study, peptide mass finger printing technique was used to identify the stress proteins (maybe anti-stress proteins for the host) extracted from B. thetaiotaomicron grown under nutrient starvation (without heme, blood or bile) prior to be placed in an aerobic solution containing a mild non-ionic detergent derived from cholic acid. We focus here on proteins related to stress, knowing that superoxide dismutase was previously identified in the extract. In parallel, the morphology of the bacterial cells was observed using electronic microscopy before and after the extraction process. The effective antioxidant effect of the extract was evaluated in vitro against hydrogen peroxide. This work highlights the B. thetaiotaomicron ability to produce a large amount of stress proteins and to remain viable during the extraction. Budding vesicles were observed on its cell wall. The extraction process did not exceed 20 h in order to preserve the bacterial viability that decreased significantly after 24 h in preliminary studies. In our experimental conditions, an inhibitory effect of the extract was found against hydrogen peroxide. Animal models of inflammation will later check in vivo if this extract of anti-stress proteins is able to counter the respiratory burst beginning an inflammation process.

Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii influence the production of mucus glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent.

BACKGROUND: The intestinal mucus layer plays a key role in the maintenance of host-microbiota homeostasis. To document the crosstalk between the host and microbiota, we used gnotobiotic models to study the influence of two major commensal bacteria, Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii, on this intestinal mucus layer. B. thetaiotaomicron is known to use polysaccharides from mucus, but its effect on goblet cells has not been addressed so far. F. prausnitzii is of particular physiological importance because it can be considered as a sensor and a marker of human health. We determined whether B. thetaiotaomicron affected goblet cell differentiation, mucin synthesis and glycosylation in the colonic epithelium. We then investigated how F. prausnitzii influenced the colonic epithelial responses to B. thetaiotaomicron. RESULTS: B. thetaiotaomicron, an acetate producer, increased goblet cell differentiation, expression of mucus-related genes and the ratio of sialylated to sulfated mucins in mono-associated rats. B. thetaiotaomicron, therefore, stimulates the secretory lineage, favoring mucus production. When B. thetaiotaomicron was associated with F. prausnitzii, an acetate consumer and a butyrate producer, the effects on goblet cells and mucin glycosylation were diminished. F. prausnitzii, by attenuating the effects of B. thetaiotaomicron on mucus, may help the epithelium to maintain appropriate proportions of different cell types of the secretory lineage. Using a mucus-producing cell line, we showed that acetate up-regulated KLF4, a transcription factor involved in goblet cell differentiation. CONCLUSIONS: B. thetaiotaomicron and F. prausnitzii, which are metabolically complementary, modulate, in vivo, the intestinal mucus barrier by modifying goblet cells and mucin glycosylation. Our study reveals the importance of the balance between two main commensal bacteria in maintaining colonic epithelial homeostasis via their respective effects on mucus.

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride.

Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.

Cytophaga-flavobacterium gliding motility.

Flavobacterium johnsoniae, like many other members of the Cytophaga-Flavobacterium-Bacteroides group, displays rapid gliding motility. Cells of F. johnsoniae glide over surfaces at rates of up to 10 microm/s. Latex spheres added to F. johnsoniae bind to and are rapidly propelled along cells, suggesting that adhesive molecules move laterally along the cell surface during gliding. Genetic analyses have identified a number of gld genes that are required for gliding. Three Gld proteins are thought to be components of an ATP-binding-cassette transporter. Five other Gld proteins are lipoproteins that localize to the cytoplasmic membrane or outer membrane. Disruption of gld genes results not only in loss of motility, but also in resistance to bacteriophages that infect wild-type cells, and loss of the ability to digest the insoluble polysaccharide chitin. Two models that attempt to incorporate the available data to explain the mechanism of F. johnsoniae gliding are presented.

The surface (S-) layer is a virulence factor of Bacteroides forsythus.

Bacteroides forsythus has emerged as a crucial periodontal pathogen with possible implications for systemic disease. The aim of this study was to isolate the S-layer from B. forsythus and examine its virulence potential as a part of efforts to characterize virulence factors of B. forsythus. The role of the S-layer in the haemagglutinating and adherent/invasive activities was evaluated. It was observed that the S-layer alone was able to mediate haemagglutination. In adherent and invasive studies, transmission electron microscopy clearly revealed that B. forsythus cells were able to attach to and invade KB cells, showing the formation of a microvillus-like extension around adherent and intracellular bacteria. The quantitative analysis showed that five different B. forsythus strains exhibited attachment (1.9-2.3 %) and invasion (0.4-0.7 %) capabilities. It was also observed through antibody inhibition assays that adherent/invasive activities of B. forsythus are mediated by the S-layer. Furthermore, an in vivo immunization study adopting a murine abscess model was used to prove that the S-layer is involved in the infectious process of abscess formation. While mice immunized with purified S-layer and B. forsythus whole cells did not develop any abscesses when challenged with viable B. forsythus cells, unimmunized mice developed abscesses. Collectively, the data obtained from these studies indicate that the S-layer of B. forsythus is a virulence factor.

A morphological and immunolabeling study of freeze-substituted Bacteroides forsythus.

We used a rapid freezing and freeze-substitution technique without glutaraldehyde and OsO4 fixation for the electron microscopic immunocytochemical demonstration of the surface structure of Bacteroides forsythus, an anaerobic Gram-negative periodontopathogen. Cells were applied to pieces of filter paper and freeze-substituted by plunge-freezing in liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR White resin. The membrane ultrastructure of B. forsythus was preserved well, and the labeling density of the freeze-submitted cells was compared to a conventional processing method. Our results show the usefulness of the freeze-substitution method for immunohistochemical studies of B. forsythus.

Bacterial filament formation, a defense mechanism against flagellate grazing, is growth rate controlled in bacteria of different phyla.

A facultatively filamentous bacterium was isolated from eutrophic lake water and was identified as Flectobacillus sp. strain MWH38 (a member of the Cytophaga-Flavobacterium-Bacteroides phylum) by comparative 16S rRNA gene sequence analysis. Filament formation by Flectobacillus sp. strain MWH38 and filament formation by Flectobacillus major, the closest known relative of strain MWH38, were studied in chemostat cultures under grazing pressure by the bacterivorous flagellate Ochromonas sp. strain DS and without predation at several growth rates. The results clearly demonstrated that filament formation by the two flectobacilli is growth rate controlled and thus independent of the presence of a predator. However, flagellate grazing positively influenced bacterial growth rates by decreasing bacterial biomass and thus indirectly stimulated filament formation. The results of investigations of cell elongation and filament formation by Comamonas acidovorans PX54 (a member of the beta subclass of the class Proteobacteria) supported the recent proposal that in this species the mechanism of filament formation is growth rate controlled. The finding that the grazing defense mechanism consisting of filament formation is growth rate controlled in the flectobacilli investigated and C. acidovorans PX54 (i.e., in bacteria belonging to divergent evolutionary phyla) may indicate that this mechanism is a phylogenetically widely distributed defense strategy against grazing.

Characterization of a Bacteroides species from human intestine that degrades glycosaminoglycans.

Polysaccharide lyases that can degrade glycosaminoglycans (GAGs) were identified in an anaerobic strain living in the human intestine. The strain was isolated from the stool of a healthy male and identified as Bacteroides sp. strain HJ-15. A detailed taxonomical study indicated the species is a strain of Bacteroides stercoris. The isolate was cultured and the polysaccharide lyase activity was partially purified. This enzyme preparation could act on GAGs containing either glucosamine or galactosamine suggesting the presence of both heparinases and chondroitinases. Various GAGs were incubated with the partially purified enzyme and the products formed were analyzed by strong anion-exchange high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. These studies demonstrated the presence of at least two types of polysaccharide lyases: heparin lyase and chondroitin sulfate lyase. The eliminative mechanism of these lyase enzymes was confirmed through the isolation of unsaturated disaccharide products. The heparin lyase acted on both heparin and acharan sulfate, a GAG recently isolated from Achatina fulica. The Bacteroides chondroitin lyase, acted on chondroitin sulfates A, B (dermatan sulfate), and C, resembling chondroitin lyase ABC. The presence of a GAG-degrading organism in human intestine may pose problems for the effective oral administration of GAG drugs.

Polaribacter gen. nov., with three new species, P. irgensii sp. nov., P. franzmannii sp. nov. and P. filamentus sp. nov., gas vacuolate polar marine bacteria of the Cytophaga-Flavobacterium-Bacteroides group and reclassification of 'Flectobacillus glomeratus' as Polaribacter glomeratus comb. nov.

Several psychrophilic, gas vacuolate strains of the Cytophage-Flavobacterium-Bacteroides (CFB) phylogenetic group were isolated from sea ice and water from the Arctic and the Antarctic. The closest taxonomically defined species by 16S rRNA sequence analysis is 'Flectobacillus glomeratus'. However, 'Flc. glomeratus' is phylogenetically distant from the Flectobacillus type species, Flc. major. On the basis of phenotypic, genotypic and 16S rRNA sequence analyses we propose a new genus, Polaribacter, with three new species, Polaribacter irgensii strain 23-P (ATCC 700398), Polaribacter franzmannii strain 301 (ATCC 700399) and Polaribacter filamentus strain 215 (ATCC 700397). P. filamentus is the type species of the genus. None of these species exhibits a cosmopolitan or bipolar distribution. This is the first taxonomic description of gas vacuolate bacteria in the CFB group. Additionally, we propose that 'Flc. glomeratus' be reclassified to the genus Polaribacter as P. glomeratus, comb. nov.

Antibacterial activities of tosufloxacin against anaerobic bacteria and the electron micrograph of its bactericidal effects.

Tosufloxacin, a quinolone, showed a broad antibacterial spectrum against gram-positive and gram-negative bacteria including anaerobic bacteria. Tosufloxacin was 4- to 8-fold more active than levofloxacin and ciprofloxacin. The MIC90 of tosufloxacin for clinical isolates of Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron and Peptostreptococcus asaccharolyticus were 0.78, 0.39, 1.56 and 0.39 micrograms/ml, respectively. Morphological observation with the scanning and transmission electron microscope revealed that exposure of B. fragilis ATCC 25285 to tosufloxacin resulted in the formation of filamentous cells with mesosome-like structures. Tosufloxacin also induced the mini-cell resulting from the unusual cell division system and a number of holes in the outer membrane. Tosufloxacin at 4 MIC caused some change in cell wall organization and cell lysis. After exposure of P. asaccharolyticus ATCC 14953 to tosufloxacin, the cells increased considerably in size and the cell wall and cross wall thickening was observed.

Immunoblot and ultrastructural analysis of antigens extracted from Dichelobacter nodosus with potassium thiocyanate.

The antigens extracted from Dichelobacter nodosus with potassium thiocyanate were analyzed by western blotting with sera from footrot-free sheep and from sheep infected with D. nodosus to identify components specific for infection. Several components with molecular mass < 33 kD were associated with infection, particularly bands of 32.5 kD, 30.5 kD, and 28 kD. Components with molecular mass > 56 kD may be responsible for false-positive reactions observed when sera of footrot-free sheep react with the potassium thiocyanate extract in solid phase enzyme-linked immunosorbent assays (ELISAs). Bands of 62.5 kD, 56 kD, 40 kD, and 19 kD were recognized for most sheep regardless of their disease status or ELISA reactivity and therefore do not appear to be detected by the ELISA. No components of the potassium thiocyanate extract were completely specific for infection. Antigens in the extract were identified primarily on the cell envelope by immunogold electron microscopy. Labeling was concentrated in the periplasm, with minor labeling of the cell surface. The extract consisted of tangled strands of particles with electron-dense cores, and few vesicular structures were observed.

Digestion of cell-wall monosaccharides of ryegrass and alfalfa hays by the ruminal bacteria Fibrobacter succinogenes and Butyrivibrio fibrisolvens.

The ruminal bacteria Fibrobacter succinogenes strains S85 and BL2 were grown in monocultures or in coculture with strain D1 of Butyrivibrio fibrisolvens, and the solubilization of ryegrass and alfalfa cell walls (CW) and digestion of CW monosaccharides were measured. Fibrobacter succinogenes monocultures and cocultures with B. fibrisolvens D1 degraded 58-69% of ryegrass CW, solubilizing 67-78% of CW glucose, 65-71% of CW xylose, 69-75% of hemicellulose, and 68-77% of total CW monosaccharides. When grown on alfalfa CW, those cultures degraded 28-39% of alfalfa CW, solubilizing 42-58% of CW glucose, 30-36% of CW xylose, and 37-45% of hemicellulose. With respect to both substrates, F. succinogenes strains solubilized CW carbohydrates better than did B. fibrisolvens D1. Complementary interaction between B. fibrisolvens D1 and the F. succinogenes strains was identified with respect to the utilization of some solubilized carbohydrates, but not with respect to the extent of CW solubilization, which was determined mainly by the F. succinogenes strains. For both substrates, utilization of cellulose by F. succinogenes monocultures was high (96-98%), whereas that of hemicellulose was lower (24-26% in ryegrass and 49-50% in alfalfa). Under scanning electron microscopy, F. succinogenes bacterial cells attached to and colonized on CW particles were characterized by the appearance of protuberant surface structures that we have identified as "polycellulosome complexes."

The effects of antigenic competition on the efficacy of multivalent footrot vaccines.

A multivalent footrot vaccine has been developed, containing pilus antigens produced in recombinant Pseudomonas aeruginosa and representing all nine serogroups of Dichelobacter (Bacteroides) nodosus commonly recognised in the field. The responses of sheep to the multivalent vaccine have been compared with those to monovalent vaccines representing only a single serogroup. Antigenic competition between serogroups occurred in sheep immunised with the multivalent formation, but high levels of protection were still achieved. The study showed that in multivalent footrot vaccines, antigenic competition is predominantly due to the presence of a family of immunologically-related pilus antigens rather than to interference by extraneous proteins.

The effect of time between doses on serological response to a recombinant multivalent pilus vaccine against footrot in sheep.

The response of sheep to a recombinant multivalent footrot vaccine containing pilus antigens was examined after the administration of two doses of vaccine at intervals ranging from 2 to 52 weeks. Agglutinating antibody titres were measured 3 weeks after the second vaccination and showed that lengthening of the interdose interval results in higher agglutinin titres. The capability of sheep to mount an increasingly strong immune response as the interval between doses is increased provides an opportunity to maximise the usefulness of vaccination by administering the first dose well before an expected footrot transmission period. This advantage of increasing the interdose interval has not been reported for traditional, whole-cell footrot vaccines, and use of the new pilus vaccine in this manner may improve prospects for disease control. Furthermore, sheep given a third dose either 6 or 12 months after their initial two-dose vaccination program achieved significantly higher titres than those elicited after the second dose, suggesting the likelihood of further improvement in disease control in successive seasons.

A preliminary study on the effects of the Nd:YAG laser on root surfaces and subgingival microflora in vivo.

The purpose of this study was to examine the effects of root preparation using the pulsed Nd:YAG laser, either alone or in combination with manual instrumentation. Study specimens consisting of 18 teeth with associated periodontal pockets from 8 different patients were treated as follows: 5 specimens were root planed with curets followed by laser exposure for 3 minutes using energy settings of 3.0 W at 20 pps; 2 specimens were root planed and then laser treated for 3 minutes using settings of 2.25 W and 20 pps; 4 specimens were treated by laser for 1 minute at settings of 1.75 W and 20 pps followed by root planing; 4 specimens were treated by laser only for 1 minute using settings of 1.75 W and 20 pps; and the remaining 3 teeth served as untreated controls. Both prior to and after completion of the laser and root planing treatments, microbiological samples were obtained from the treated pockets and submitted to a commercial laboratory for analysis of levels of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia. With the exception of two 7-day specimens, all others were extracted immediately post-therapy and processed for SEM examination. All treated specimens, regardless of treatment sequence, exhibited some degree of laser-induced root surface alteration. Notably, laser-treated calculus deposits were free of their characteristic surface layer of microbial plaque. Microbial sampling indicated a post-therapy reduction in levels of all 3 putative microbial pathogens compared to pre-treatment samples and control specimens. However, SEM examination revealed residual deposits of plaque and calculus in all treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Description of Porphyromonas circumdentaria sp. nov. and reassignment of Bacteroides salivosus (Love, Johnson, Jones, and Calverley 1987) as Porphyromonas (Shah and Collins 1988) salivosa comb. nov.

A new species, Porphyromonas circumdentaria, is proposed for pigmented, asaccharolytic strains that were isolated from the gingival margins or mouth-associated diseases of cats. This bacterium is an obligately anaerobic, gram-negative, brown- or black-pigmented, asaccharolytic, nonmotile, nonsporing, rod-shaped organism which does not grow in bile and has a guanine-plus-cytosine content of 40 to 42 mol%. It produces major amounts of acetic, butyric, and isovaleric acids and minor amounts of propionic, isobutyric, and phenylacetic acids as end products of metabolism in cooked meat medium. Glutamate and malate dehydrogenases are present, while 6-phosphogluconate and glucose-6-phosphate dehydrogenases are absent. The major cellular fatty acid is 13-methyltetradecanoic acid (iso-C15:0 acid). P. circumdentaria strains are catalase positive and produce ammonia, and colonies fluoresce under short-wavelength UV light. These strains do not hemagglutinate erythrocytes, exhibit trypsinlike activity, or produce chymotrypsin or alpha-fucosidase. They are heavily piliated and produce a capsule. The type strain is strain VPB 3329 (= NCTC 12469). Bacteroides salivosus (D. N. Love, J. L. Johnson, R. F. Jones, and A. Calverley, Int. J. Syst. Bacteriol. 37:307-309, 1987) is an obligately anaerobic, gram-negative, pigmented, asaccharolytic, nonmotile, rod-shaped organism which does not grow in bile and has a guanine-plus-cytosine content of 42 to 44 mol%. This organism produces major amounts of acetic, butyric, and phenylacetic acids and minor amounts of isobutyric and isovaleric acids as end products of metabolism in cooked meat medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Further studies on some physical and biochemical characteristics of asaccharolytic pigmented Bacteroides of feline origin.

The ultrastructure of the appendages of 24 strains of asaccharolytic pigmented Bacteroides spp. of cats was studied by transmission electron microscopy. All strains examined by thin section showed abundant fimbriae, outer membrane vesicles and capsules. Negative staining showed fimbriae which varied from long, fine and wavy in Bact. salivosus and cat Group 2 to shorter, less abundant and thicker fimbriae in cat strains of Bact. gingivalis as well as type strains of Porphyromonas gingivalis and P. asaccharolytica. Capsular material was very thick amorphous in human P. gingivalis, cat strains of Bact. gingivalis and in P. assaccharolytica but fine and fibrillary in preparations of Bact. salivosus and cat Group 2 organisms. Wet india ink preparations showed a large capsule although those of Bact. salivosus and Group 2 appeared largest. Five-day Group 2 broth cultures featured a thick ropy growth consistent with a large accumulation of extracellular slime. Enzymatic activities of the 24 strains measured by API ZYM system as well as the conventional biochemical tests showed it was possible to differentiate reliably Bact. salivosus from the other cat strains of asaccharolytic pigmented Bacteroides species and from human P. gingivalis and P. endodontalis by a combination of these tests. These tests suggest that Bact. salivosus is unlikely to belong to the genus Prevotella. Its place within the genus Porphyromonas is still to be determined.