• Journal of Korean society of Dental Hygiene
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• v.6 no.4
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• pp.311-324
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• 2006

The purpose of this investigation was to evaluate of the specificity of Fusobacterium nucleatum subspecies-specific DNA probes using dot blot hybridization. To confirm whether the clinical isolates were F. nucleatum or not, 16S rDNA of them were cloned and sequenced. The sequencing data were used in homology search with database of GenBank. When the homology was above 98% compared with the nucleotide sequence of a certain bacteria, it was judged as the same species with the bacteria. 23 strains of F. nucleatum were isolates from subgingival plaque of periodontitis patient. The clinical isolates of F. nucleatum were classified into 10 groups using phylogenetic analysis of 16S rDNA sequence. F. nucleatum subspecies nucleatum-specific DNA probe Fu4(1.3 kb) reacted with genomic DNAs from 8 type strains of F. nucleatum and it reacted strongly with those from 8 clinical isolates. The Fp4(0.8 kb) reacted with F. nucleatum subsp. polymorphum ATCC 10953 and one clinical isolates. Fv35(1.9 kb) and Fs17(8.2 kb) probes reacted with genomic DNAs from F. nucleatum subsp. vincentii ATCC 49256 and F. nucleatum subsp. fusiform ATCC 51190, respectively. Our results showed that it is not enough to evaluate the specificity of F. nucleatum subspecies-specific DNA probes with only dot blot hybridization. Therefore, Southern blot analysis will be necessary to confirm the specificity of F. nucleatum subspecies-specific DNA probes.

• Journal of Microbiology
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• v.43 no.4
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• pp.331-336
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• 2005

The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC $25586^T$ (F. nucleatum ATCC $25586^T$), and to develop sets of strain-specific polymerase chain reaction (PCR) primers. Strain-specificity was tested against 16 strains of F. nucleatum and 3 strains of distinct Fusobacterium species. Southern blot hybridization revealed that the Fu12 reacted exclusively with the HindIII-digested genomic DNA of F. nucleatum ATCC $25586^T$. The results of PCR revealed that three pairs of PCR primers, based on the nucleotide sequence of Fu12, generated the strain-specific amplicons from F. nucleatum ATCC $25586^T$. These results suggest that the DNA probe Fu12 and the three pairs of PCR primers could be useful in the identification of F. nucleatum ATCC $25586^T$, especially with regard to the determination of the authenticity of the strain.

• International Journal of Oral Biology
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• v.30 no.2
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• pp.59-63
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• 2005

Postantibiotic effects (PAE) refer to suppression of the bacterial growth following limited periods of exposure to an antibiotic and subsequent to the removal of the antibiotic agent. Fusobacterium nucleatum and Porphyromonas gingivalis are Gram-negative anaerobic bacteria associated with several periodontal diseases. In this study, postantibiotic effects (PAE), postantibiotic sub-MIC effect (PA SME) and sub-MIC effect (SME) of antibiotics on F. nucleatum ATCC 25586 and P. gingivalis W50 were investigated. The PAE was induced by 10X the MIC of antibiotic and antibiotic was eliminated by washing. The PA SMEs were studied by addition of 0.1, 0.2 and 0.3X MICs during the postantibiotic phase of the bacteria, and the SMEs were studied by exposition of the bacteria to antibiotic at the sub-MICs only. Amoxicillin, doxycycline and tetracycline induced PAE for F. nucleatum ATCC 25586 and P. gingivalis W50. But metronidazole and penicillin induced PAE for only F. nucleatum ATCC 25586. Metronidazole and doxycycline induced PA SME and SME for both species of anaerobic bacteria used in this study. The PA SME values for both strains were substantially longer than the SME values. The present study showed the existence of PAE, PA SME and SME for various antibiotics against F. nucleatum ATCC 25586 and P. gingivalis W50.

• International Journal of Oral Biology
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• v.45 no.2
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• pp.70-75
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• 2020

The aim of this study was to identify strain KCOM 1265 isolated from subgingival plaque at the species level by comparing 16S ribosomal RNA gene (16S rDNA) and genome sequences. The whole genome of strain KCOM 1265 was extracted using the phenol-chloroform extraction method. 16S rDNA was amplified using polymerase chain reaction and sequenced using the dideoxy chain termination method. Pairwise genome comparison was performed using average nucleotide identity (ANI) and genome-to-genome distance (GGD) analyses. The data showed that the percent similarity of 16S rDNA sequence of strain KCOM 1265 was 99.6% as compared with those of Fusobacterium polymorphum ATCC 10953^T and Fusobacterium hwasookii KCOM 1249^T. The ANI values of strain KCOM 1265 with F. polymorphum ATCC 10953^T and F. hwasookii KCOM 1249^T were 95.8% and 93.0%, respectively. The GGD values of strain KCOM 1265 with F. polymorphum ATCC 10953^T and F. hwasookii KCOM 1249^T were 63.9% and 49.6%, respectively. These results indicate that strain KCOM 1265 belongs to F. polymorphum.

• Restorative Dentistry and Endodontics
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• v.14 no.1
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• pp.85-96
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• 1989

In the Infected root canals, the majority of the isolated bacteria are either strict anaerobes or microaerophilic organisms. Among thease, Black-pigmented Bacteroides species are the most important and have relation with clinical symptoms. The purpose of this study were to investigate on the 7 different types of bacteria which include Black-pigmented Bacteroides - Black--pigmented Bacteroides, Actinobacillus actinomycetemcomitans, Streptococcus, Capnocytophaga, Eiknella corrodens, Fusobacterium, Actinomyces - and the interrelationship between these 7 bacterical species, and to compare Black-pigmented Bacteroides with the clinical symptoms in infected root canals. The canal contents of 15 necrotized teeth with 8 clinical symptoms were sampled and cultured aerobically and anaerobically for growth in 7 selective agar plates for 7 bacterial species. Black-pigmented Bacteroides and Streptococcus subspecies were identified by biochemical tests. The results were as follows; 1. 70.51% of the bacteria isolated were anaerobes and 29.49% were aerobes. 2. B. loescheii, B. intermedius, B. denticola were isolated in 8 cases, Streptococcus was isolated in 8 cases, Fusobacterium was isolated in 6 cases, Actinomyces was isolated in 5 cases and Eiknella corrodens, Actinobacillus actinomycetemcomitans, Capnocytophaga were not isolated. 3. There were mutual inhibition of growth between Black-pigmented Bacteroides and Streptococcus. (P<0.01) But Actinomyces and Fusobacterium showed mutual aids for growth. (P<0.05) 4. Black-Pigmented Bacteroides was found to be related both to sinus tract formation and to apical sensitivity to palpation.(P < 0.05).

• Korean Journal of Microbiology
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• v.12 no.3
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• pp.131-137
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• 1974

It is a well-known fact that an isolation of non-sporeforming anaerobes, considered normal flora in man ordinarily but causes serious infections sometimes, is a dificult procedure because of their great oxygen sensitivity. Among the many techniques employed in clinical laboratories, despite of its high expenses, the GasPak method has been most widely used because of its relative simplicity. On the other hand, the steel wool method has gained a good reputation recently. This technique makes it possible to treat individual plate so that any single specimen can be promptly cultured anaerobically. The procedure is quite simple and the expenses are negligible. In the present study it is to compare these two methods as to their efficiency of anaerobic cultivation using 13 VPI strains of non-sporeforming amaerobic bacteria. Among the 13 species the following 11, Bacteroides fragilis ss. fragilis, B. fragilis ss. thetaiotaomicron, propionibacterium acnes, Eubacterium limosum, E. lentum, peptococcus asaccharolyticus, Pc. prevotii, Pc. magnus, peptostreptococcus anaerobius, Ps. intermedius nad Veillonella parvula, grew well with the steel wool method whose colony numbers reaching 57 to 119% of those with GasPak method. The remaining two species, Fusobacterium nucleatum and F.necrophorum, did not grow well with the steel wool method showing the colony numbers were only 0.4% of those with GasPak method in the case of Fusobacterium nucleatum. In the case of Fusobacterium necrophorum, very few colonies developed even with a heavy inoculation. As to the size of colonies, there were no significant difference between these two methods.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.519-524
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• 2005

An ${\alpha}$-L-rhamnosidase (EC 3.2.1.40.), which transforms quercitrin to quercetin, was purified from Fusobacterium K-60, a human intestinal anaerobic bacterium. The specific activity of the purified ${\alpha}$-L-rhamnosidase was 2.89 mol/min/mg protein. ${\alpha}$-L-Rhamnosidase, whose molecular size was 170 kDa by gel filtration, was composed of four subunits ($M_r$ 41,000 Da) with pI and optimal pH values of 5.2 and 5.5-7.0, respectively. The apparent $K_m$ and $V_{max}$ values for p-nitrophenyl-${\alpha}$-L-rhamnopyranoside and quercitrin were determined to be 0.057 mM and 3.4 mol/min/mg, and 0.077 mM and 5.0 mol/min/mg, respectively. This enzyme was strongly inhibited by $Cu^{2+},\;Mn^{2+}$, L-rhamnose, and p-chlormercuriphenylsulfonic acid. These findings suggest that the biochemical properties and substrate specificity of the purified enzyme are different from those of the previously purified ${\alpha}$-L-rhamnosidase. This is the first reported purification of quercitrin-hydrolyzing ${\alpha}$-L-rhamnosidase from intestinal bacteria.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.859-865
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• 2008

Bacterial glycosyltransferases have drawn growing attention as economical enzymes for oligosaccharide synthesis, with their easy expression and relatively broad substrate specificity. Here, we characterized a glycosyltransferase homolog (Fnu_GT) from a human oral pathogen, Fusobacterium nucleatum. Bioinformatic analysis showed that Fnu_GT belongs to the glycosyltransferases family II. The recombinant Fnu_GT (rFnu_GT) expressed in Escherichia coli displayed the highest glycosylation activity when UDP-galactose (Gal) was used as a donor nucleotide-sugar with heptose or N-acetylglucosamine (GlcNAc) as an acceptor sugar. Interestingly, rFnu_GT transferred the galactose moiety of UDP-Gal to a nonreducing terminal GlcNAc attached to the trimannosyl core glycan, indicating its potential as an enzyme for human-type N-glycan synthesis.

• International Journal of Oral Biology
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• v.46 no.3
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• pp.127-133
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• 2021

We previously showed that γ-glutamyltranspeptidase (GGT), an enzyme involved in glutathione metabolism, in Bacillus subtilis acts as a virulence factor for osteoclastogenesis via the RANKL-dependent pathway. Hence, it can be hypothesized that GGT of periodontopathic bacteria acts as a virulence factor in bone destruction. Because Fusobacterium nucleatum, which is a periodontopathic pathogen, has GGT with a primary structure similar to that of B. subtilis GGT (37.7% identify), the bone-resorbing activity of F. nucleatum GGT was examined here. Recombinant GGT (rGGT) of F. nucleatum was expressed in Escherichia coli and purified using the His tag of rGGT. F. nucleatum rGGT (Fn rGGT) was expressed as a precursor of GGT, and then processed to a heavy subunit and a light subunit, which is characteristic of general GGTs, including the human and B. subtilis enzymes. Osteoclastogenesis was achieved in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. Fn rGGT induced osteoclastogenesis to a level similar to that of B. subtilis rGGT; furthermore, osteoclastogenesis was induced in a dose-dependent manner. These results suggest that F. nucleatum GGT possesses a virulent bone-resorbing activity, which could play an important role in the pathogenesis of periodontitis.

• Genomics & Informatics
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• v.21 no.2
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• pp.21.1-21.14
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• 2023

Fusobacterium nucleatum is a gram-negative bacteria associated with diverse infections like appendicitis and colorectal cancer. It mainly attacks the epithelial cells in the oral cavity and throat of the infected individual. It has a single circular genome of 2.7 Mb. Many proteins in F. nucleatum genome are listed as "Uncharacterized." Annotation of these proteins is crucial for obtaining new facts about the pathogen and deciphering the gene regulation, functions, and pathways along with discovery of novel target proteins. In the light of new genomic information, an armoury of bioinformatic tools were used for predicting the physicochemical parameters, domain and motif search, pattern search, and localization of the uncharacterized proteins. The programs such as receiver operating characteristics determine the efficacy of the databases that have been employed for prediction of different parameters at 83.6%. Functions were successfully assigned to 46 uncharacterized proteins which included enzymes, transporter proteins, membrane proteins, binding proteins, etc. Apart from the function prediction, the proteins were also subjected to string analysis to reveal the interacting partners. The annotated proteins were also put through homology-based structure prediction and modeling using Swiss PDB and Phyre2 servers. Two probable virulent factors were also identified which could be investigated further for potential drug-related studies. The assigning of functions to uncharacterized proteins has shown that some of these proteins are important for cell survival inside the host and can act as effective drug targets.