Draft Genome Sequence of Fusobacterium nucleatum subsp. fusiforme ATCC 51190T

Fusobacterium nucleatum, one of the major causative bacteria of periodontitis, is classified into five subspecies (nucleatum, polymorphum, vincentii, animalis, and fusiforme) on the basis of the several phenotypic characteristics and DNA homology. This is the first report of the draft genome sequence of F. nucleatum subsp. fusiforme ATCC 51190(T).     

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.     

Efficient gene transfer and targeted mutagenesis in Fusobacterium nucleatum

Fusobacterium nucleatum is a Gram-negative anaerobe important in dental biofilm ecology and infectious diseases with significant societal impact. The lack of efficient genetic systems has hampered molecular analyses in this microorganism. We previously reported construction of a shuttle plasmid, pHS17, using the native fusobacterial plasmid pFN1 and an erythromycin resistance cassette. However, the host range of pHS17 was restricted to F. nucleatum, ATCC 10953, and the transformation efficiency was limited. This study was undertaken to improve genetic systems for molecular analysis in F. nucleatum. We identified a second F. nucleatum strain, ATCC 23726, which is transformed with improved efficiency compared to ATCC 10953. Two novel second generation pFN1-based shuttle plasmids, pHS23 and pHS30, were developed and enable transformation of ATCC 23726 at 6.2 x 10(4) and 1.5 x 10(6) transformants/mug plasmid DNA, respectively. The transformation efficiency of pHS30, which harbors a catP gene conferring resistance to chloramphenicol, was more than 1000-fold greater than that of pHS17. The improved transformation efficiency facilitated disruption of the chromosomal rnr gene using a suicide plasmid pHS19, the first demonstration of targeted mutagenesis in F. nucleatum. These results provide significant advances in the development of systems for molecular analysis in F. nucleatum.     

Protein secretion systems in Fusobacterium nucleatum: genomic identification of Type 4 piliation and complete Type V pathways brings new insight into mechanisms of pathogenesis

Recent genomic analyses of the two sequenced strains F. nucleatum subsp. nucleatum ATCC 25586 and F. nucleatum subsp. vincentii ATCC 49256 suggested that the major protein secretion systems were absent. However, such a paucity of protein secretion systems is incongruous with F. nucleatum pathogenesis. Moreover, the presence of one or more such systems has been described for every other Gram-negative organism sequenced to date. In this investigation, the question of protein secretion in F. nucleatum was revisited. In the current study, the absence in F. nucleatum of a twin-arginine translocation system (TC #2.A.64.), a Type III secretion system (TC #3.A.6.), a Type IV secretion system (TC #3.A.7.) and a chaperone/usher pathway (TC #1.B.11.) was confirmed. However, contrary to previous findings, our investigations indicated that a Type I protein secretion system was also absent from F. nucleatum. In contrast, members of the holin family (TC #1.E) and the machinery required for a Type 4 piliation/fimbriation system (TC #3.A.15.2.) were identified using a variety of bioinformatic tools. Furthermore, a complete range of proteins resembling members of the Type V secretion pathway, i.e., the Type Va (autotransporter; TC #1.B.12.), Type Vb (two-partner secretion system; TC #1.B.20.) and Type Vc (YadA-like trimeric autotransporter; TC #1.B.42.), was found. This work provides new insight into the protein secretion and virulence mechanisms of F. nucleatum.     

Intrageneric relationships of members of the genus Fusobacterium as determined by reverse transcriptase sequencing of small-subunit rRNA.

The phylogenetic interrelationships of 14 members of the genus Fusobacterium were investigated by performing a comparative analysis of the 16S rRNA sequences of these organisms. The sequence data revealed considerable intrageneric heterogeneity. The four species Fusobacterium nucleatum (including F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, "F. nucleatum subsp. fusiforme," and "F. nucleatum subsp. animalis"), Fusobacterium alocis, Fusobacterium periodonticum, and Fusobacterium simiae, which colonize oral cavities, exhibited high levels of sequence homology with each other and formed a distinct group within the genus. Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans also formed a phylogenetically coherent group, as did the two species Fusobacterium gonidiaformans and Fusobacterium necrophorum. Fusobacterium russii and Fusobacterium necrogenes displayed no specific relationship with any of the other fusobacteria. The sequence data are discussed in the context of previous physiological and chemical findings.     

Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum

The major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fev1. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36.5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.     

Heterogeneity within Fusobacterium nucleatum, proposal of four subspecies

Fusobacterium nucleatum strains, isolated from man and animals, were shown to comprise four centres of variation within the species by using a variety of biochemical tests. DNA-DNA hybridization data indicated that they should differences between the groups to warrant their placement into four subspecies for which we propose the following: F. nucleatum subsp. nucleatum (commonly isolated from diseased sites), F. nucleatum subsp. polymorphum (from healthy sites, most frequently isolated), F. nucleatum subsp. fusiforme (from healthy sites, most frequently isolated), F. nucleatum subsp. fusiforme (from healthy sites, rarely isolated) and F. nucleatum subsp. animalis from the colon of animals.     

Fluorescence based measurements of Fusobacterium nucleatum coaggregation and of fusobacterial attachment to mammalian cells

Fusobacterium nucleatum is a common oral anaerobe associated with gingivitis, periodontal disease and preterm deliveries. Coaggregation among oral bacteria is considered to be a significant factor in dental plaque development. Adhesion to host cells was suggested to be important for the F. nucleatum virulence associated with oral inflammation and with preterm births. An uncharacterized fusobacterial galactose inhibitible adhesin mediates coaggregation of F. nucleatum 12230 and F. nucleatum PK1594 with the periodontal pathogen Porphyromonas gingivalis. This adhesin is also involved with the attachment of both fusobacterial strains to host cells. However, it has been suggested that additional unidentified fusobacterial adhesins are involved in F. nucleatum virulence associated with preterm births. In this study, a fluorescence-based high throughput sensitive and reproducible method was developed for measuring bacterial coaggregation and bacterial attachment to mammalian cells. Using this method we found that coaggregation of F. nucleatum 4H with P. gingivalis and its attachment to murine macrophages is less inhibitible by galactose than that of F. nucleatum PK1594. These findings suggest that F. nucleatum 4H can serve as a model organism for identifying nongalactose inhibitible F. nucleatum adhesins considered to be involved in fusobacterial attachment to mammalian cells.     

Genotypic and phenotypic characterization of fusobacteria from Chinese and European patients with inflammatory periodontal diseases

Phylogenetic and antigenic studies were performed on 48 human oral Fusobacterium strains from Chinese patients with either necrotizing ulcerative gingivitis (NUG) or gingivitis and on 23 Fusobacterium nucleatum or Fusobacterium periodonticum strains from European periodontitis patients. Alignment of partial 16S rRNA gene sequences resulted in a phylogenetic tree that corresponded well with the current classification of oral fusobacteria into F. periodonticum and several subspecies of F. nucleatum, in spite of much minor genetic variability. F. periodonticum, F. nucleatum subsp. animalis and a previously undescribed phylogenetic cluster (C4), that may represent an additional F. nucleatum subspecies, constituted discrete clusters distinct from the remainder of F. nucleatum with high bootstrap values. Chinese and European strains differed markedly with regard to their respective classification patterns, suggesting a predominance of F. peridonticum and F. nucleatum susp. animalis over F. nucleatum subsp. nucleatum and F. nucleatum subsp. fusiforme/vincentii in samples from China. Antigenic typing enabled the association of many previously described serovars with distinct phylogenetic clusters and when applied directly to uncultured clinical samples confirmed the differential distribution of oral Fusobacterium taxa in Chinese and European samples. Bacteria from cluster C4 and F. nucleatum subsp. animalis were significantly more prevalent and accounted for higher cell numbers in NUG than in gingivitis samples, suggesting a possible association of these rarely observed taxa with NUG in Chinese patients.     

Native plasmids of Fusobacterium nucleatum: characterization and use in development of genetic systems

Three native plasmids of Fusobacterium nucleatum were characterized, including DNA sequence analysis of one plasmid, pFN1. A shuttle plasmid, pHS17, capable of transforming Escherichia coli and F. nucleatum ATCC 10953 was constructed with pFN1. pHS17 was stably maintained in the F. nucleatum transformants, and differences in the transformation efficiencies suggested the presence of a restriction-modification system in F. nucleatum.     

Diisopropylfluorophosphate-binding proteins in the outer membrane of Fusobacterium nucleatum: strain variations

Six strains of Fusobacterium nucleatum were tested for their ability to react with [3H]diisopropylfluorophosphate (DFP), a serine protease inhibitor. Several cytoplasmic proteins were labelled but the strongest labelling was regularly observed in a few outer membrane proteins. The number and the molecular mass of the proteins detected varied according to the strain tested. A 61 kDa protein was labelled in all strains tested, including the two type strains ATCC 10953 and ATCC 25586. A 65 kDa protein was particularly strongly labelled in strains Fev1 and F6.     

Development of strain-specific PCR primers based on a DNA probe Fu12 for the identification of fusobacterium nucleatum subsp. nucleatum ATCC 25586T

The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC 25586T (F. nucleatum ATCC 25586T), 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 25586T. 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 25586T. 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 25586T, especially with regard to the determination of the authenticity of the strain.     

Identification and characterization of a novel adhesin unique to oral fusobacteria

Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.     

Survey of chimeric IStron elements in bacterial genomes: multiple molecular symbioses between group I intron ribozymes and DNA transposons

IStrons are chimeric genetic elements composed of a group I intron associated with an insertion sequence (IS). The group I intron is a catalytic RNA providing the IStron with self-splicing ability, which renders IStron insertions harmless to the host genome. The IS element is a DNA transposon conferring mobility, and thus allowing the IStron to spread in genomes. IStrons are therefore a striking example of a molecular symbiosis between unrelated genetic elements endowed with different functions. In this study, we have conducted the first comprehensive survey of IStrons in sequenced genomes that provides insights into the distribution, diversity, origin and evolution of IStrons. We show that IStrons have a restricted phylogenetic distribution limited to two bacterial phyla, the Firmicutes and the Fusobacteria. Nevertheless, diverse IStrons representing two major groups targeting different insertion site motifs were identified. This taken with the finding that while the intron components of all IStrons belong to the same structural class, they are fused to different IS families, indicates that multiple intron–IS symbioses have occurred during evolution. In addition, introns and IS elements related to those that were at the origin of IStrons were also identified.     

Bifidobacterium animalis subsp. lactis ATCC 27673 Is a Genomically Unique Strain within Its Conserved Subspecies

Many strains of Bifidobacterium animalis subsp. lactis are considered health-promoting probiotic microorganisms and are commonly formulated into fermented dairy foods. Analyses of previously sequenced genomes of B. animalis subsp. lactis have revealed little genetic diversity, suggesting that it is a monomorphic subspecies. However, during a multilocus sequence typing survey of Bifidobacterium, it was revealed that B. animalis subsp. lactis ATCC 27673 gave a profile distinct from that of the other strains of the subspecies. As part of an ongoing study designed to understand the genetic diversity of this subspecies, the genome of this strain was sequenced and compared to other sequenced genomes of B. animalis subsp. lactis and B. animalis subsp. animalis. The complete genome of ATCC 27673 was 1,963,012 bp, contained 1,616 genes and 4 rRNA operons, and had a G+C content of 61.55%. Comparative analyses revealed that the genome of ATCC 27673 contained six distinct genomic islands encoding 83 open reading frames not found in other strains of the same subspecies. In four islands, either phage or mobile genetic elements were identified. In island 6, a novel clustered regularly interspaced short palindromic repeat (CRISPR) locus which contained 81 unique spacers was identified. This type I-E CRISPR-cas system differs from the type I-C systems previously identified in this subspecies, representing the first identification of a different system in B. animalis subsp. lactis. This study revealed that ATCC 27673 is a strain of B. animalis subsp. lactis with novel genetic content and suggests that the lack of genetic variability observed is likely due to the repeated sequencing of a limited number of widely distributed commercial strains.     

Lateral gene transfer in Mycobacterium avium subspecies paratuberculosis

Lateral gene transfer is an integral part of genome evolution in most bacteria. Bacteria can readily change the contents of their genomes to increase adaptability to ever-changing surroundings and to generate evolutionary novelty. Here, we report instances of lateral gene transfer in Mycobacterium avium subsp. paratuberculosis, a pathogenic bacteria that causes Johne's disease in cattle. A set of 275 genes are identified that are likely to have been recently acquired by lateral gene transfer. The analysis indicated that 53 of the 275 genes were acquired after the divergence of M. avium subsp. paratuberculosis from M. avium subsp. avium, whereas the remaining 222 genes were possibly acquired by a common ancestor of M. avium subsp. paratuberculosis and M. avium subsp. avium after its divergence from the ancestor of M. tuberculosis complex. Many of the acquired genes were from proteobacteria or soil dwelling actinobacteria. Prominent among the predicted laterally transferred genes is the gene rsbR, a possible regulator of sigma factor, and the genes designated MAP3614 and MAP3757, which are similar to genes in eukaryotes. The results of this study suggest that like most other bacteria, lateral gene transfers seem to be a common feature in M. avium subsp. paratuberculosis and that the proteobacteria contribute most of these genetic exchanges.     

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.     

Genes of cyanobacterial origin in plant nuclear genomes point to a heterocyst-forming plastid ancestor

Plastids are descended from a cyanobacterial symbiosis which occurred over 1.2 billion years ago. During the course of endosymbiosis, most genes were lost from the cyanobacterium's genome and many were relocated to the host nucleus through endosymbiotic gene transfer (EGT). The issue of how many genes were acquired through EGT in different plant lineages is unresolved. Here, we report the genome-wide frequency of gene acquisitions from cyanobacteria in 4 photosynthetic eukaryotes--Arabidopsis, rice, Chlamydomonas, and the red alga Cyanidioschyzon--by comparison of the 83,138 proteins encoded in their genomes with 851,607 proteins encoded in 9 sequenced cyanobacterial genomes, 215 other reference prokaryotic genomes, and 13 reference eukaryotic genomes. The analyses entail 11,569 phylogenies inferred with both maximum likelihood and Neighbor-Joining approaches. Because each phylogenetic result is dependent not only upon the reconstruction method but also upon the site patterns in the underlying alignment, we investigated how the reliability of site pattern generation via alignment affects our results: if the site patterns in an alignment differ depending upon the order in which amino acids are introduced into multiple sequence alignment--N- to C-terminal versus C- to N-terminal--then the phylogenetic result is likely to be artifactual. Excluding unreliable alignments by this means, we obtain a conservative estimate, wherein about 14% of the proteins examined in each plant genome indicate a cyanobacterial origin for the corresponding nuclear gene, with higher proportions (17-25%) observed among the more reliable alignments. The identification of cyanobacterial genes in plant genomes affords access to an important question: from which type of cyanobacterium did the ancestor of plastids arise? Among the 9 cyanobacterial genomes sampled, Nostoc sp. PCC7120 and Anabaena variabilis ATCC29143 were found to harbor collections of genes which are-in terms of presence/absence and sequence similarity-more like those possessed by the plastid ancestor than those of the other 7 cyanobacterial genomes sampled here. This suggests that the ancestor of plastids might have been an organism more similar to filamentous, heterocyst-forming (nitrogen-fixing) representatives of section IV recognized in Stanier's cyanobacterial classification. Members of section IV are very common partners in contemporary symbiotic associations involving endosymbiotic cyanobacteria, which generally provide nitrogen to their host, consistent with suggestions that fixed nitrogen supplied by the endosymbiont might have played an important role during the origin of plastids.     

Genetic diversity within the genus Francisella as revealed by comparative analyses of the genomes of two North American isolates from environmental sources

ABSTRACT: BACKGROUND: Francisella tularensis is an intracellular pathogen that causes tularemia in humans and the public health importance of this bacterium has been well documented in recent history. Francisella philomiragia, a distant relative of F. tularensis, is thought to constitute an environmental lineage along with Francisella novicida. Nevertheless, both F. philomiragia and F. novicida have been associated with human disease, primarily in immune-compromised individuals. To understand the genetic relationships and evolutionary contexts among different lineages within the genus Francisella, the genome of Francisella spp. strain TX07-7308 was sequenced and compared to the genomes of F. philomiragia strains ATCC 25017 and 25015, F. novicida strain U112, and F. tularensis strain Schu S4. RESULTS: The size of strain ATCC 25017 chromosome was 2,045,775 bp and contained 1,983 protein-coding genes. The size of strain TX07-7308 chromosome was 2,035,931 bp and contained 1,980 protein-coding genes. Pairwise BLAST comparisons indicated that strains TX07-7308 and ATCC 25017 contained 1700 protein coding genes in common. NUCmer analyses revealed that the chromosomes of strains TX07-7308 and ATCC 25017 were mostly collinear except for a few gaps, translocations, and/or inversions. Using the genome sequence data and comparative analyses with other members of the genus Francisella (e.g., F. novicida strain U112 and F. tularensis strain Schu S4), several strain-specific genes were identified. Strains TX07-7308 and ATCC 25017 contained an operon with six open reading frames encoding proteins related to enzymes involved in thiamine biosynthesis that was absent in F. novicida strain U112 and F. tularensis strain Schu S4. Strain ATCC 25017 contained an operon putatively involved in lactose metabolism that was absent in strain TX07-7308, F. novicida strain U112, and F. tularensis strain Schu S4. In contrast, strain TX07-7308 contained an operon putatively involved in glucuronate metabolism that was absent in the genomes of strain ATCC 25017, F. novicida strain U112, and F. tularensis strain Schu S4. The polymorphic nature of polysaccharide biosynthesis/modification gene clusters among different Francisella strains was also evident from genome analyses. CONCLUSIONS: From genome comparisons, it appeared that genes encoding novel functions have contributed to the metabolic enrichment of the environmental lineages within the genus Francisella. The inability to acquire new genes coupled with the loss of ancestral traits and the consequent reductive evolution may be a cause for, as well as an effect of, niche selection of F. tularensis. Sequencing and comparison of the genomes of more isolates are required to obtain further insights into the ecology and evolution of different species within the genus Francisella.