Introduction

Fusobacterium nucleatum is Gram-negative, non-motile, obligately anaerobic rod bacteria, which is frequently isolated from the oral cavity and may play an important role in periodontal disease [1, 2]. Based on the PAGE pattern of the whole-cell proteins and DNA homology [3] or electrophoretic patterns of glutamate dehydrogenase and 2-oxoglutarate reductase and DNA–DNA hybridization patterns [4], F. nucleatum was classified into five subspecies (nucleatum, polymorphum, vincentii, animalis, and fusiforme). Previous studies have suggested that F. nucleatum subsp. vincentii is genetically similar to F. nucleatum subsp. fusiforme [5, 6]. Therefore, this study was designed to investigate the possibility of classifying these two subspecies into a single subspecies by phylogenetic analysis of 22 housekeeping genes: rpoB (DNA-dependent RNA polymerase beta-subunit), atpC (F0F1-type ATP synthase gamma subunit), exoA (exodeoxyribonuclease III), pfkA (6-phosphofructokinase), pyrB (aspartate carbamoyltransferase), gapA (glyceraldehyde 3-phosphate dehydrogenase), pgk (phosphoglycerate kinase), gpmA (phosphoglycerate mutase), pckA (phosphoenolpyruvate carboxykinase), ldh (l-lactate dehydrogenase), ung (uracil-DNA glycosylase), tpi (triosephosphate isomerase), oadA (oxaloacetate decarboxylase alpha subunit), ddl (d-alanine-d-alanine ligase), murA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase), eno (phosphopyruvate hydratase), dnaB (DNA helicase), pgi (glucose-6-phosphate isomerase), recA (recombinase A), gyrA (DNA gyrase subunit A), gyrB (DNA gyrase subunit B), and groEL (molecular chaperone GroEL) for phylogenetic analysis of the five F. nucleatum subspecies type strains. Table 1 showed the GenBank accession numbers and analyzed nucleotide location of the 22 housekeeping genes.

Table 1 Information of the nucleotide sequences of the genes used in this study
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A 24,715-bp single sequence was generated by integrating individual genes in the mentioned order. Phylogenetic analysis was conducted using MEGA version 5 [7]. Phylogenetic trees were constructed using the neighbor-joining method and bootstrap analysis was carried out using 1,000 replications. Sequence similarities were calculated using MegAlign program (Lasergene™ 8.0, DNAStar, Inc., Madison, WI, USA).

The phylogenetic tree showed that all of the F. nucleatum strains were clearly separated into four distinct clusters with 100 % bootstrap value (Fig. 1a). The genome sequence of eight strains used in this study has been deposited in GenBank. According to a dendrogram generated by the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/genome?term=Fusobacterium%20nucleatum; NIH, Bethesda, MD, USA), F. nucleatum subsp. vincentii ATCC 49256T and F. nucleatum subsp. fusiforme ATCC 51190T were clustered in the same group and each strain of other F. nucleatum subspecies were also separated into the same cluster. Our data is consistent with that of NCBI except for the similarity between the four clusters composed of each F. nucleatum subspecies strain, including the one consisting of F. nucleatum subsp. vincentii ATCC 49256T and F. nucleatum subsp. fusiforme ATCC 51190T. The sequence similarity of the concatenated 22 housekeeping genes between F. nucleatum subsp. vincentii ATCC 49256T and F. nucleatum subsp. fusiforme ATCC 51190T was 98.5 % (Fig. 1b). The sequence similarities between the strains of the others F. nucleatum subspecies ranged from 98.3 to 99.5 %. The phylogenetic tree also delineated the F. nucleatum groups from Fusobacterium periodonticum (Fig. 1a). The sequences similarities between F. nucleatum strains and F. periodonticum ATCC 33369T ranged from 88.9 to 90.1 % (mean 89.4 %) (Fig. 1b). This is in agreement with our previous report, in that F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme could not be distinguished from one another based on the analyses of the sequences of 16S rRNA gene, rpoB, and the zinc protease gene [6]. These findings suggest that these two subspecies 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 considered as a later synonym of F. nucleatum subsp. vincentii Dzink et al. 1990.

Fig. 1
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a Phylogenetic tree based on a single sequence (24,715 bp) by concatenating 22 housekeeping genes of eight Fusobacterium nucleatum strains, including five type strains of F. nucleatum subspecies. The resulting tree topology was evaluated by bootstrap analyses of the neighbor-joining tree based on 1,000 replications using MEGA version 5 [7]. b Percentage identity among the eight strains of F. nucleatum and F. polymorphum ATCC 33369T. Sequence similarities were calculated using the MegAlign program (DNAStar Lasergene™ 8.0, DNAStar, Inc.)

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