Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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Genomic heterogeneity in a natural archaeal population suggests a model of tRNA gene disruption

Sugahara J, Fujishima K, Nunoura T, Takaki Y, Takami H, Takai K, Tomita M, Kanai A.

PLoS ONE (PLoS ONE) 7 ( 3 ) 1-8 2012.03

Research paper (scientific journal), Joint Work

Understanding the mechanistic basis of the disruption of tRNA genes, as manifested in the intron-containing and split tRNAs found in Archaea, will provide considerable insight into the evolution of the tRNA molecule. However, the evolutionary processes underlying these disruptions have not yet been identified. Previously, a composite genome of the deep-branching archaeon Caldiarchaeum subterraneum was reconstructed from a community genomic library prepared from a C. subterraneum-dominated microbial mat. Here, exploration of tRNA genes from the library reveals that there are at least three types of heterogeneity at the tRNA Thr(GGU) gene locus in the Caldiarchaeum population. All three involve intronic gain and splitting of the tRNA gene. Of two fosmid clones found that encode tRNA Thr(GGU), one (tRNA Thr-I) contains a single intron, whereas another (tRNA Thr-II) contains two introns. Notably, in the clone possessing tRNA Thr-II, a 5′ fragment of the tRNA Thr-I (tRNA Thr-F) gene was observed 1.8-kb upstream of tRNA Thr-II. The composite genome contains both tRNA Thr-II and tRNA Thr-F, although the loci are >500 kb apart. Given that the 1.8-kb sequence flanked by tRNA Thr-F and tRNA Thr-II is predicted to encode a DNA recombinase and occurs in six regions of the composite genome, it may be a transposable element. Furthermore, its dinucleotide composition is most similar to that of the pNOB8-type plasmid, which is known to integrate into archaeal tRNA genes. Based on these results, we propose that the gain of the tRNA intron and the scattering of the tRNA fragment occurred within a short time frame via the integration and recombination of a mobile genetic element. © 2012 Sugahara et al.

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Bioinformatics Tools for Mass Spectroscopy-Based Metabolomic Data Processing and Analysis

Sugimoto, Masahiro; Kawakami, Masato; Robert, Martin; Soga, Tomoyoshi; Tomita, Masaru

Current Bioinformatics (Current Bioinformatics) 7 ( 1 ) 96-108 - 108 2012.03

Research paper (scientific journal), Joint Work, ISSN 15748936

Biological systems are increasingly being studied in a holistic manner, using omics approaches, to provide quantitative and qualitative descriptions of the diverse collection of cellular components. Among the omics approaches, metabolomics, which deals with the quantitative global profiling of small molecules or metabolites, is being used extensively to explore the dynamic response of living systems, such as organelles, cells, tissues, organs and whole organisms, under diverse physiological and pathological conditions. This technology is now used routinely in a number of applications, including basic and clinical research, agriculture, microbiology, food science, nutrition, pharmaceutical research, environmental science and the development of biofuels. Of the multiple analytical platforms available to perform such analyses, nuclear magnetic resonance and mass spectrometry have come to dominate, owing to the high resolution and large datasets that can be generated with these techniques. The large multidimensional datasets that result from such studies must be processed and analyzed to render this data meaningful. Thus, bioinformatics tools are essential for the efficient processing of huge datasets, the characterization of the detected signals, and to align multiple datasets and their features. This paper provides a state-of-the-art overview of the data processing tools available, and reviews a collection of recent reports on the topic. Data conversion, pre-processing, alignment, normalization and statistical analysis are introduced, with their advantages and disadvantages, and comparisons are made to guide the reader. © 2012 Bentham Science Publishers.

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Measures of Compositional Strand Bias Related to Replication Machinery and its Applications

Arakawa K., Tomita M.

Current Genomics (Current Genomics) 13 ( 1 ) 4 - 15 2012.03

ISSN 13892029

The compositional asymmetry of complementary bases in nucleotide sequences implies the existence of a mutational or selectional bias in the two strands of the DNA duplex, which is commonly shaped by strand-specific mechanisms in transcription or replication. Such strand bias in genomes, frequently visualized by GC skew graphs, is used for the computational prediction of transcription start sites and replication origins, as well as for comparative evolutionary genomics studies. The use of measures of compositional strand bias in order to quantify the degree of strand asymmetry is crucial, as it is the basis for determining the applicability of compositional analysis and comparing the strength of the mutational bias in different biological machineries in various species. Here, we review the measures of strand bias that have been proposed to date, including the ΔGC skew, the B 1 index, the predictability score of linear discriminant analysis for gene orientation, the signal-to-noise ratio of the oligonucleotide bias, and the GC skew index. These measures have been predominantly designed for and applied to the analysis of replication-related mutational processes in prokaryotes, but we also give research examples in eukaryotes. ©2012 Bentham Science Publishers.

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Metatranscriptomic Analysis of Microbes in an Oceanfront Deep-Subsurface Hot Spring Reveals Novel Small RNAs and Type-Specific tRNA Degradation

Murakami S., Fujishima K., Tomita M., Kanai A.

Applied and Environmental Microbiology (Applied and Environmental Microbiology) 78 ( 4 ) 1015-1022 - 1022 2012.02

Research paper (scientific journal), Joint Work, ISSN 00992240

Studies of small noncoding RNAs (sRNAs) have been conducted predominantly using culturable organisms, and the acquisition of further information about sRNAs from global environments containing uncultured organisms now is very important. In this study, hot spring water (57°C, pH 8.1) was collected directly from the underground environment at depths of 250 to 1,000min Yunohama, Japan, and small RNA sequences obtained from the environment were analyzed. A phylogenetic analysis of both archaeal and bacterial 16S rRNA gene sequences was conducted, and the results suggested the presence of unique species in the environment, corresponding to the Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN) group and three new Betaproteobacteria. A metatranscriptomic analysis identified 64,194 (20,057 nonredundant) cDNA sequences. Of these cDNAs, 90% were either tRNAs, tRNA fragments, rRNAs, or rRNA fragments, whereas 2,181 reads (10%) were classified as previously uncharacterized putative candidate sRNAs. Among these, 15 were particularly abundant, 14 of which showed no sequence similarity to any known noncoding RNA, and at least six of which form very stable RNA secondary structures. The analysis of a large number of tRNA fragments suggested that unique relationships exist between the anticodons of the tRNAs and the sites of tRNA degradation. Previous bacterial tRNA degradation studies have been limited to specific organisms, such as Escherichia coli and Streptomyces coelicolor, and the current results suggest that specific tRNA decay occurs more frequently than previously expected. © 2012, American Society for Microbiology.

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MMMDB: Mouse multiple tissue metabolome database

Sugimoto M., Ikeda S., Niigata K., Tomita M., Sato H., Soga T.

Nucleic Acids Research (Nucleic Acids Research) 40 ( D1 ) D809 - 14 2012.01

ISSN 03051048

The Mouse Multiple Tissue Metabolome Database (MMMDB) provides comprehensive and quantitative metabolomic information for multiple tissues from single mice. Manually curated databases that integrate literature-based individual metabolite information have been available so far. However, data sets on the absolute concentration of a single metabolite integrated from multiple resources are often difficult to be used when different metabolomic studies are compared because the relative balance of the multiple metabolite concentrations in the metabolic pathways as a snapshot of a dynamic system is more important than the absolute concentration of a single metabolite. We developed MMMDB by performing non-targeted analyses of cerebra, cerebella, thymus, spleen, lung, liver, kidney, heart, pancreas, testis and plasma using capillary electrophoresis time-of-flight mass spectrometry and detected 428 non-redundant features from which 219 metabolites were successfully identified. Quantified concentrations of the individual metabolites and the corresponding processed raw data; for example, the electropherograms and mass spectra with their annotations, such as isotope and fragment information, are stored in the database. MMMDB is designed to normalize users' data, which can be submitted online and used to visualize overlaid electropherograms. Thus, MMMDB allows newly measured data to be compared with the other data in the database. MMMDB is available at: http:// mmmdb.iab.keio.ac.jp. © The Author(s) 2011.

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Time-structure of the yeast metabolism in vivo

Sasidharan K., Tomita M., Aon M., Lloyd D., Murray D.B.

Advances in Experimental Medicine and Biology 2012

ISSN 00652598

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Metabolomic profiling rationalized pyruvate efficacy in cybrid cells harboring MELAS mitochondrial DNA mutations.

Kami, K., Fujita, Y., Igarashi, S., Koike, S., Sugawara, S., Ikeda, S., Sato, N., Ito, M., Tanaka, M., Tomita, M. and Soga, T.

Mitochondrion. (Mitochondrion) 12 ( 6 ) 644 - 653 2012

Accepted, ISSN 15677249

Pyruvate treatment was found to alleviate clinical symptoms of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome and is highly promising therapeutic. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we measured time-changes of 161 intracellular and 85 medium metabolites to elucidate metabolic effects of pyruvate treatment on cybrid human 143B osteosarcoma cells harboring normal (2SA) and MELAS mutant (2SD) mitochondria. The results demonstrated dramatic and sustainable effects of pyruvate administration on the energy metabolism of 2SD cells, corroborating pyruvate as a metabolically rational treatment regimen for improving symptoms associated with MELAS and possibly other mitochondrial diseases. © 2012 Elsevier B.V. and Mitochondria Research Society.

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Metabolic profiling reveals new serum biomarkers for differentiating diabetic nephropathy

Hirayama, A., Nakashima, E., Sugimotoa, M., Akiyama, S., Sato, W., Maruyama, S., Matsuo, S., Tomita, M., Yuzawa, Y. and Soga, T.

Anal Bioanal Chem (Analytical and Bioanalytical Chemistry) 404 ( 10 ) 3101-9 - 3109 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 16182642

Capillary electrophoresis coupled with time-offlight mass spectrometry was used to explore new serum biomarkers with high sensitivity and specificity for diabetic nephropathy (DN) diagnosis, through comprehensive analysis of serum metabolites with 78 diabetic patients. Multivariate analyses were used for identification of marker candidates and development of discriminative models. Of the 289 profiled metabolites, orthogonal partial leastsquares discriminant analysis identified 19 metabolites that could distinguish between DN with macroalbuminuria and diabetic patients without albuminuria. These identified metabolites included creatinine, aspartic acid, γ-butyrobetaine, citrulline, symmetric dimethylarginine (SDMA), kynurenine, azelaic acid, and galactaric acid. Significant correlations between all these metabolites and urinary albumin-to-creatinine ratios (p<0.009, Spearman's rank test) were observed. When five metabolites (including γ-butyrobetaine, SDMA, azelaic acid and two unknowns) were selected from 19 metabolites and applied for multiple logistic regressionmodel, AUC value for diagnosing DN was 0.927 using the whole dataset, and 0.880 in a cross-validation test. In addition, when four known metabolites (aspartic acid, SDMA, azelaic acid and galactaric acid) were applied, the resulting AUC was still high at 0.844 with the whole dataset and 0.792 with cross-validation. Combination of serum metabolomics with multivariate analyses enabled accurate discrimination of DN patients. The results suggest that capillary electrophoresis-mass spectrometry based metabolome analysis could be used for DN diagnosis. © Springer-Verlag 2012.

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A simple method to provide a shuttling plasmid for delivery to other host ascertained by prolonged stability of extracellular plasmid DNA released from Escherichia coli K12 endA mutant, deficient in major endonuclease

Itaya, M.,　Kawata, Y., Sato, M., Tomita, M. and Nakahigashi, K.

J. Biochem. (Journal of Biochemistry) 152 ( 6 ) 501–504 - 504 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021924X

Escherichia coli lyses by lambda phage propagation. Circular plasmid DNA was present during E. coli lysis as an extracellular plasmid DNA (excpDNA) that was stable enough to transform coexisting competent Bacillus subtilis cells. Detailed investigations unveiled that excpDNA is transient in both quality and quantity, with stability lasting no more than several hours. A survey using E. coli lambda lysogens with various genetic backgrounds demonstrated that the loss of Endonuclease I (ΔendA::kan) conferred extraordinary stability upon excpDNA for as long as 48 h. Studies on endA mutants suggested that excpDNA remained localized in cell debris, in contrast to E. coli genome DNA, which diffused into medium at an early point in lysis. Lambda lysogens constructed on endA recA mutants are presented for potential pipelines in delivery to other competent proficient microbes. © 2012 The Authors.

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A medium-chain fatty acid as an alternative energy source in mouse preimplantation development

Yamada, M., Takanashi, K., Hamatani, T., Hirayama, A., Akutsu, H., Fukunaga, T., Ogawa, S., Sugawara, K., Shinoda, K., Soga, T., Umezawa, A., Kuji, N., Yoshimura, Y. and Tomita, M.

Sci Rep. (Scientific Reports) 2 930 2012

Research paper (scientific journal), Joint Work, Accepted

To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for β-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-13 C 8[ octanoate added to the culture media into intermediate metabolites of the TCA cycle via β-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

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Light Microscopy and Phylogenetic Analyses of Chlamydomonas Species (Volvocales, Chlorophyceae). : I. Chlamydomonas pseudomacrostigma and Chlamydomonas media

Nakada, T., Nozaki, H. and Tomita, M.

Acta phytotaxonomica et geobotanica 62:69-78 62 69 - 78 2012

Joint Work

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Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: a BAC-based domino method applied to DNA with a high GC content

Ohtani, N., Hasegawa, M., Sato, M., Tomita, M., Kaneko, S. and Itaya, M.

Biotechnol J. (Biotechnology Journal) 7 ( 7 ) 867 - 76 2012

Joint Work, Accepted, ISSN 18606768

Bacillus subtilis is the only bacterium-based host able to clone giant DNA above 1000 kbp. DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. To expand the target DNA range to higher GC content, we tried to clone a pTT27 megaplasmid (257 kbp, 69% of G+C) from Thermus thermophilus. To facilitate the reconstruction process, we subcloned pTT27 in a bacterial artificial chromosome (BAC) vector of Escherichia coli. Owing to the ability of BAC to carry around 100 kbp DNA, only 4 clones were needed to cover the pTT27 and conduct step-by-step assembly in the B. subtilis genome. The full length of 257 kbp was reconstructed through 3 intermediary lengths (108, 153, and 226 kbp), despite an unexpected difficulty in the maintenance of DNA >200 kbp. Retrieval of these four pTT27 segments from the B. subtilis genome by genetic transfer to a plasmid pLS20 was attempted. A stable plasmid clone was obtained only for the 108 and 153 kbp intermediates. The B. subtilis genome was demonstrated to accommodate large DNA with a high GC content, but may be restricted to less than 200 kbp by unidentified mechanisms. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Metabolomics: A New Omics Technology for Traditional Herbal Medicine Analysis

Iino, K., Sugimoto, M., Soga, T. and Tomita, M.

Chinese Medicine: Acupuncture, Herbal Medicine and Therapies 45-55 2012

Research paper (scientific journal), Joint Work, Accepted

Peptide Identification by Searching Large-Scale Tandem Mass Spectra against Large Databases: Bioinformatics Methods in Proteogenomics

Helmy, M, Tomita M, Ishihama Y

Genes, Genome and Genomics 6 76-85 2012

Research paper (scientific journal), Joint Work, Accepted

Profiling of the charged metabolites of traditional herbal medicines using capillary electrophoresis time-of-flight mass spectrometry

Iino¸ K., Sugimoto¸ M., Soga¸ T. and Tomita, M.

Metabolomics 8 ( 1 ) 99-108 - 108 2012

Research paper (scientific journal), Joint Work, ISSN 15733882

The quantification of a small number of bioactive components in herbal medicines is often inadequate when attempting to elucidate a medicine's biological effects. Despite rapid advances in analytical technologies, obtaining comprehensive metabolomic profiles of herbal medicines remains difficult, due to the complexity of natural product mixtures. Toki-Shakuyaku-San is a Chinese medicine used widely to treat gynecological and obstetric disorders, such as infertility, dysmenorrhea, toxemia during pregnancy and neural dysfunction. It consists of Angelica acutiloba Radix (Toki), Cnidium officinale Rhizoma (Senkyu), Paeonia lactiflora Radix (Shakuyaku), Atractylodes lancea Rhizoma (Sojutsu), Alisma orientale Rhizoma (Takusha) and Poria cocos Hoelen (Bukuryo). To elucidate the composition of these herbal medicines individually, we conducted non-targeted profiling analyses of extracts of these herbs using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which allows the simultaneous quantification of hundreds of charged metabolites. In total, 737 ± 183. 1 (average ± SD) metabolite-derived features were observed, and of these, 119 metabolites were identified. Score plots of principal component analysis (PCA) showed a clear cluster including Shakuyaku, Bukuryo, and Sojutsu, while the other three herbs were distributed over PCA spaces. Loading plots revealed that amino acids and shikimate-derived alkaloids were the predominant metabolite constituents. Hierarchical clustering analysis revealed that few clusters overlapped in the herbal medicines tested. This report is the first demonstration of the characterization of a herbal medicine using large-scale metabolomic analysis, which is complementary to traditional quality control methods. © 2011 Springer Science+Business Media, LLC.

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Validation of Bacterial Replication Termination Models Using Simulation of Genomic Mutations

Kono, N., Arakawa, K.,and Tomita, M.

PLoS ONE (PLoS ONE) 7 ( 4 ) e34526 2012

Research paper (scientific journal), Joint Work, Accepted

In bacterial circular chromosomes and most plasmids, the replication is known to be terminated when either of the following occurs: the forks progressing in opposite directions meet at the distal end of the chromosome or the replication forks become trapped by Tus proteins bound to Ter sites. Most bacterial genomes have various polarities in their genomic structures. The most notable feature is polar genomic compositional asymmetry of the bases G and C in the leading and lagging strands, called GC skew. This asymmetry is caused by replication-associated mutation bias, and this "footprint" of the replication machinery suggests that, in contrast to the two known mechanisms, replication termination occurs near the chromosome dimer resolution site dif. To understand this difference between the known replication machinery and genomic compositional bias, we undertook a simulation study of genomic mutations, and we report here how different replication termination models contribute to the generation of replication-related genomic compositional asymmetry. Contrary to naive expectations, our results show that a single finite termination site at dif or at the GC skew shift point is not sufficient to reconstruct the genomic compositional bias as observed in published sequences. The results also show that the known replication mechanisms are sufficient to explain the position of the GC skew shift point. © 2012 Kono et al.

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Nematode-specific tRNAs that decode an alternative genetic code for leucine

Hamashima, K., Fujishima, K., Masuda, T., Sugahara, J., Tomita, M. and Kanai, A.

Nucleic Acids Research (Nucleic Acids Research) 40 ( 8 ) 3653-3662 - 3662 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 03051048

Class II transfer RNAs (tRNAs), including tRNALeu and tRNA Ser, have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them 'nematode-specific V-arm-containing tRNAs' (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNALeu. In vitro aminoacylation assays showed that nev-tRNAGly and nev-tRNAIle are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNA Gly decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes. © 2011 The Author(s).

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The role of Staphylococcal enterotoxin in atopic keratoconjunctivitis and corneal ulceration

Fujishima, H., Okada, N., Dogru., M., Baba, F., Tomita, M., Abe, J., Matsumoto, K. and Saito, H.

Allergy 67 ( 6 ) 799-803 - 803 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 01054538

Background Patients with atopic eczema frequently experience colonization with Staphylococcus aureus that is directly correlated with the eczema severity. We hypothesized that S. aureus-secreted enterotoxins (SE) are involved in the pathophysiology of atopic keratoconjunctivitis (AKC). Methods A total of 45 subjects (18 with AKC, nine vernal keratoconjunctivitis (VKC), eight seasonal allergic conjunctivitis (SAC), and ten healthy volunteers) were enrolled. Slit lamp examinations, including fluorescein staining, were performed. Scraped samples were collected from the upper tarsal conjunctiva, lower conjunctival sacs, and the skin around the eyelid margins. Superantigen (SAg) genes were detected using polymerase chain reaction (PCR). Results Among 45 cases, S. aureus was detected significantly more in AKC patients than VKC patients (P = 0.026), SAC patients (P = 0.0003), and healthy volunteers (P = 0.0001). SAg genes were detected in 11 patients. SEB (2/11), SEG (8/11), and SEI (8/11) were detected, but no other SE. There was a significant difference in SE detection between AKC and SAC patients (P = 0.03). In severe types of ocular allergic disease such as AKC and VKC (N = 27), SE was detected in six of ten patients with corneal ulcers and two of 17 patients without corneal ulcers. SE was detected in significantly more patients with corneal ulcers (P = 0.025). Conclusions In patients with AKC, S. aureus and SE were detected more frequently compared with other patients and healthy volunteers, especially in association with corneal ulceration suggesting a role of SE. So far, it is unknown whether SE leads to tissue damage of the cornea by initiating an immune response or has direct toxic effects. © 2012 John Wiley & Sons A/S.

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Systems Biology, Metabolomics, and Cancer Metabolism

Tomita, M.,Kami, K.

Science 336 990-991 2012

Research paper (scientific journal), Joint Work, Accepted

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The third plasmid pVV8 from Thermus thermophilus HB8: isolation, characterization, and sequence determination

Ohtani., N., Tomita, M. and Itaya, M.

Extremophiles (Extremophiles) 16 ( 2 ) 237-44 - 244 2012

Research paper (scientific journal), Joint Work, ISSN 14310651

The extremely thermophilic bacterium Thermus thermophilus is a model organism for structural biology and systems biology, and the so-called "Structural and Functional Whole-Cell Project for T. thermophilus HB8" is in progress. The released genomic sequence of the strain HB8 is composed of chromosome, pTT27 megaplasmid, and pTT8 plasmid. In this paper, however, a third plasmid was demonstrated and its sequence was determined. Although this plasmid pVV8 had been reported before, limited information and an unfortunate dropout in the substrain, whose genomic sequence was determined, would have prevented the plasmid from coming to public attention. The intrinsic circular plasmid, which was estimated to be six to ten copies in a cell, is 81151 bp and its G + C content is 68%. Among the identified 91 ORFs, a single gene has been experimentally analyzed before and is known as xylose isomerase. The phnCDEGHIJKLMX operon related to phosphonate metabolism, alkaline phosphatase, putative transcriptional regulators, several sets of toxin-antitoxin system, and transposase-like ORFs are also encoded on the pVV8 plasmid. Although association with cell aggregation was the one phenotypic characteristic of the plasmid that had been reported, it was never confirmed. Comparison of T. thermophilus HB8 strains suggests that the pVV8 is nonessential for growth. © 2011 Springer.

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