Masahito Fukunaga

Borrelia tanukii Sp. Nov. and Borrelia turdae Sp. Nov. Found from Ixodid Ticks in Japan: Rapid Species Identification by 16S rRNA Gene-Targeted PCR Analysis

Based on the results of RFLP‐ribotyping, whole DNA/DNA hybridization and phylogenetic analysis of the 16S rRNA gene, we previously defined two genomic groups of spirochetes closely related to Borrelia burgdorferi sensu lato: group Hk501 for strains isolated from Ixodes tanuki ticks and group Ya501 for strains isolated from Ixodes turdus ticks. In this report, we propose that group Hk501 should be classified as Borrelia tanukii sp. nov. and group Ya501 as Borrelia turdae sp. nov. The alignment of previously published Borrelia 16S rRNA gene sequences led us to design species‐specific PCR primer sets. The primers allowed the rapid identification of B. tanukii and B. turdae.

Mitochondrial genetic code in cestodes

The flatworm mitochondrial genetic code, which has been used for all species of the Platyhelminthes, is mainly characterized by AUA codon for isoleucine, AAA codon for asparagine and UAA codon for tyrosine. In eight species of cestodes (Echinococcus multilocularis, Echinococcus granlosus, Taenia solium Taenia saginata, Taenia hydatigena, Taenia crassiceps, Hymenolepis nama and Mesocestoides corti), the cytochrome c oxidase subunit I (COI) genes were partially sequenced to verify this genetic code. Comparison of the COI-encoding nucleotide sequences with those of human, sea urchin, fruit fly, nematode and yeast indicated that the assignments of AUA and AAA codons are adequate for cestodes. In addition, the nucleotide sequences of ATPase subunit 6 (ATP6) gene and its flanking region were compared to examine initiation and stop codons. In the related species of T. solium and T. saginata, the deduced amino acid sequences of ATP6 were homogeneous; however, the conversion of initiation codon AUG into GUG was observed in T. saginata. We also found the similar conversion in T. crassiceps. The C-terminal sequences of putative ATP6 proteins were highly conserved among the eight species and the stop codon UAG was altered to UAA in all Taenia species. The features of the gene-junctional region between NADH dehydrogenase subunit 4 (ND4) and glutamine tRNA (tRNAGln) genes also supported that UAA serves as a stop codon. Based on these results, we propose that the flatworm mitochondrial code should be modified for cestodes, particularly, in an initiating methionine codon (GUG) and a terminating codon (UAA).

The complete mitochondrial DNA sequence of the cestode Echinococcus multilocularis (Cyclophyllidea: Taeniidae).

The 13,738 bp mitochondrial DNA from the cestode Echinococcus multilocularis has been sequenced. It contains two major noncoding regions and 36 genes (12 for proteins involved in oxidative phosphorylation, two for rRNAs and 22 for tRNAs) but a gene for ATPase subunit 8 is missing. All genes are transcribed in the same direction. Putative secondary structures of tRNAs indicate that most of them are conventional clover leaves but the dihydrouridine arm is unpaired in tRNA(Ser(AGN)), tRNA(Ser(UCN)), tRNA(Arg) and tRNA(Cys). The base composition at the wobble positions of fourfold degenerate codon families is highly biased toward U and against C.

Novel Mitochondrial Gene Content and Gene Arrangement Indicate Illegitimate Inter-mtDNA Recombination in the Chigger Mite, Leptotrombidium pallidum

To better understand the evolution of mitochondrial (mt) genomes in the Acari (mites and ticks), we sequenced the mt genome of the chigger mite, Leptotrombidium pallidum (Arthropoda: Acari: Acariformes). This genome is highly rearranged relative to that of the hypothetical ancestor of the arthropods and the other species of Acari studied. The mt genome of L. pallidum has two genes for large subunit rRNA, a pseudogene for small subunit rRNA, and four nearly identical large noncoding regions. Nineteen of the 22 tRNAs encoded by this genome apparently lack either a T-arm or a D-arm. Further, the mt genome of L. pallidum has two distantly separated sections with identical sequences but opposite orientations of transcription. This arrangement cannot be accounted for by homologous recombination or by previously known mechanisms of mt gene rearrangement. The most plausible explanation for the origin of this arrangement is illegitimate inter-mtDNA recombination, which has not been reported previously in animals. In light of the evidence from previous experiments on recombination in nuclear and mt genomes of animals, we propose a model of illegitimate inter-mtDNA recombination to account for the novel gene content and gene arrangement in the mt genome of L. pallidum.

A Phylogenetic Analysis of Saccharomyces Species by the Sequence of 18S–28S rRNA Spacer Regions

Sequences of two internally transcribed spacer regions between 18S and 28S rRNA genes were determined to assess the phylogenetic relationship in the strains belonging to the genus Saccharomyces. The sequences of S. bayanus and S. pastorianus were quite similar, but not identical. Two phylogenetic trees constructed by the neighbor‐joining method showed that all the species examined were distinguished from one another. The Saccharomyces sensu stricto species: S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus, were closely related and far from the Saccharomyces sensu lato species including S. barnetti, S. castellii, S. dairensis, S. exiguus, S. servazzii, S. spencerorum and S. unisporus, and an outlying species, S. kluyveri.

Treponema medium sp. nov., Isolated from Human Subgingival Dental Plaque

A new Treponema species, for which we propose the name Treponema medium, was isolated from subgingival plaque from an adult with periodontal disease. The morphological characteristics, differential biochemical characteristics, and protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of this organism are described. The guanine-plus-cytosine content of the DNA of T. medium is 51 mol%. The levels of DNA-DNA relatedness of the new species to other Treponema species, including Treponema denticola, Treponema vincentii, Treponema socranskii, Treponema pallidum, and Treponema phagedenis, are less than 30%. A phylogenetic analysis based on 16S rRNA sequences distinguished the new Treponema strain from strains belonging to previously described Treponema species. The type strain of T. medium is strain G7201.

Molecular Mechanisms for the Variation of Mitochondrial Gene Content and Gene Arrangement Among Chigger Mites of the Genus Leptotrombidium (Acari: Acariformes)

The gene content of a mitochondrial (mt) genome, i.e., 37 genes and a large noncoding region (LNR), is usually conserved in Metazoa. The arrangement of these genes and the LNR is generally conserved at low taxonomic levels but varies substantially at high levels. We report here a variation in mt gene content and gene arrangement among chigger mites of the genus Leptotrombidium. We found previously that the mt genome of Leptotrombidium pallidum has an extra gene for large-subunit rRNA (rrnL), a pseudo-gene for small-subunit rRNA (PrrnS), and three extra LNRs, additional to the 37 genes and an LNR typical of Metazoa. Further, the arrangement of mt genes of L. pallidum differs drastically from that of the hypothetical ancestor of the arthropods. To find to what extent the novel gene content and gene arrangement occurred in Leptotrombidium, we sequenced the entire or partial mt genomes of three other species, L. akamushi, L. deliense, and L. fletcheri. These three species share the arrangement of all genes with L. pallidum, except trnQ (for tRNA-glutamine). Unlike L. pallidum, however, these three species do not have extra rrnL or PrrnS and have only one extra LNR. By comparison between Leptotrombidium species and the ancestor of the arthropods, we propose that (1) the type of mt genome present in L. pallidum evolved from the type present in the other three Leptotrombidium species, and (2) three molecular mechanisms were involved in the evolution of mt gene content and gene arrangement in Leptotrombidium species.

Borrelia recurrentis characterization and comparison with relapsing-fever, Lyme-associated, and other Borrelia spp.

Borrelia recurrentis, the cause of louse-borne relapsing fever, has until recently been considered noncultivable, which has prevented characterization of this spirochete. We successfully cultivated 18 strains from patients with louse-borne relapsing fever and present the initial characterization of these isolates. Electron microscopy revealed spirochetal cells with pointed ends, an average wavelength of 1.8 microns, an amplitude of 0.8 micron, and 8 to 10 periplasmic flagella. The G+C ratio was 28.4 mol%. Whole DNA-DNA hybridizations showed similarity between the isolates of B. recurrentis but not with Borrelia hermsii, Borrelia parkeri, Borrelia turicatae, or the Lyme-associated borreliae. Sequencing studies of both the flagellin and 16S RNA genes revealed that the greatest similarity was between B. recurrentis and Borrelia duttonii. Analysis of the sodium dodecyl sulfate-polycarylamide gel electrophoresis profiles of strains revealed four groups based on the position of a major protein band (one of the groups showed some heterogeneity and was subdivided into four subgroups). Pulsed-field gel electrophoresis revealed five distinct patterns.

The mitochondrial genomes of soft ticks have an arrangement of genes that has remained unchanged for over 400 million years

There are two major groups of ticks: soft ticks and hard ticks. The hard ticks comprise the prostriate ticks and the metastriate ticks. The mitochondrial (mt) genomes of one species of prostriate tick and two species of metastriate ticks had been sequenced prior to our study. The prostriate tick has the ancestral arrangement of mt genes of arthropods, whereas the two metastriate ticks have rearrangements of eight genes and duplicate control regions. However, the arrangement of genes in the mt genomes of soft ticks had not been studied. We sequenced the mt genomes of two species of soft ticks, Carios capensis and Ornithodoros moubata, and a metastriate tick, Haemaphysalis flava. We found that the soft ticks have the ancestral arrangement of mt genes of arthropods, whereas the metastriate tick, H. flava, shares the rearrangements of mt genes and duplicate control regions with the other two metastriate ticks that have previously been studied. Our study indicates that gene rearrangements and duplicate control regions in mt genomes occurred once in the most recent common ancestor of metastriate ticks, whereas the ancestral arrangement of arthropods has remained unchanged for over 400 million years in the lineages leading to the soft ticks and the prostriate ticks.

Successful in vitro cultivation of Borrelia duttonii and its comparison with Borrelia recurrentis.

Borrelia duttonii, the cause of East African tick-borne relapsing fever, has until now been refractory to growth in laboratory media. This spirochaete has only be propagated in mice or by tissue culture, restricting both yield and purity of cells available for research. The successful isolation of five clinical isolates of B. duttonii from patients in Central Tanzania and their comparison with Borrelia recurrentis is reported. Electron microscopy revealed spirochaetal cells with pointed ends, a mean wavelength of 1.8 microns with an amplitude of 0.8 micron, similar to the findings for B. recurrentis. Cells contained 10 periplasmic flagella inserted at each end of the spirochaete, again comparable with the counts of 8-10 flagella found in B. recurrentis. PFGE revealed a chromosome of approximately 1 Mb, a large plasmid of approximately 200 kb, and a small plasmid of 11 kb in all strains of B. duttonii and in B. recurrentis. B. duttonii possessed a further 7-9 plasmids with sizes ranging from 20 to 90 kb. In two isolates of B. duttonii, the profiles were identical. In contrast, all 18 isolates of B. recurrentis fell into one of five plasmid patterns with 3-4 plasmids ranging from 25 to 61.5 kb in addition to those of 11 and 200 kb described above. Analysis of the SDS-PAGE profiles of B. duttonii strains revealed a high-molecular-mass band of 33.4-34.2 kDa in four strains (variable large protein, VLP) and a low-molecular-mass band of 22.3 kDa in the remaining strain (variable small protein, VSP). This resembles the protein profiles found in B. recurrentis. The G + C ratio of B. duttonii was 27.6 mol%. Nucleotide sequence of the rrs gene (16S rRNA) from four B. duttonii isolates revealed 100% identity among these strains and 99.7% homology with three strains deposited by others in GenBank. The rrs gene of eight representative clinical isolates of B. recurrentis confirmed their close similarity with B. duttonii.