Minenosuke Matsutani

Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus

Acetobacter species have been used for brewing traditional vinegar and are known to have genetic instability. To clarify the mutability, Acetobacter pasteurianus NBRC 3283, which forms a multi-phenotype cell complex, was subjected to genome DNA sequencing. The genome analysis revealed that there are more than 280 transposons and five genes with hyper-mutable tandem repeats as common features in the genome consisting of a 2.9-Mb chromosome and six plasmids. There were three single nucleotide mutations and five transposon insertions in 32 isolates from the cell complex. The A. pasteurianus hyper-mutability was applied for breeding a temperature-resistant strain grown at an unviable high-temperature (42°C). The genomic DNA sequence of a heritable mutant showing temperature resistance was analyzed by mutation mapping, illustrating that a 92-kb deletion and three single nucleotide mutations occurred in the genome during the adaptation. Alpha-proteobacteria including A. pasteurianus consists of many intracellular symbionts and parasites, and their genomes show increased evolution rates and intensive genome reduction. However, A. pasteurianus is assumed to be a free-living bacterium, it may have the potentiality to evolve to fit in natural niches of seasonal fruits and flowers with other organisms, such as yeasts and lactic acid bacteria.

Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses.

BackgroundHigh-temperature fermentation technology with thermotolerant microbes has been expected to reduce the cost of bioconversion of cellulosic biomass to fuels or chemicals. Thermotolerant Kluyveromyces marxianus possesses intrinsic abilities to ferment and assimilate a wide variety of substrates including xylose and to efficiently produce proteins. These capabilities have been found to exceed those of the traditional ethanol producer Saccharomyces cerevisiae or lignocellulose-bioconvertible ethanologenic Scheffersomyces stipitis.ResultsThe complete genome sequence of K. marxianus DMKU 3-1042 as one of the most thermotolerant strains in the same species has been determined. A comparison of its genomic information with those of other yeasts and transcriptome analysis revealed that the yeast bears beneficial properties of temperature resistance, wide-range bioconversion ability, and production of recombinant proteins. The transcriptome analysis clarified distinctive metabolic pathways under three different growth conditions, static culture, high temperature, and xylose medium, in comparison to the control condition of glucose medium under a shaking condition at 30°C. Interestingly, the yeast appears to overcome the issue of reactive oxygen species, which tend to accumulate under all three conditions.ConclusionsThis study reveals many gene resources for the ability to assimilate various sugars in addition to species-specific genes in K. marxianus, and the molecular basis of its attractive traits for industrial applications including high-temperature fermentation. Especially, the thermotolerance trait may be achieved by an integrated mechanism consisting of various strategies. Gene resources and transcriptome data of the yeast are particularly useful for fundamental and applied researches for innovative applications.

Complete Genome Sequence of NBRC 3288, a Unique Cellulose-Nonproducing Strain of Gluconacetobacter xylinus Isolated from Vinegar

Gluconacetobacter xylinus is involved in the industrial production of cellulose. We have determined the genome sequence of G. xylinus NBRC 3288, a cellulose-nonproducing strain. Comparative analysis of genomes of G. xylinus NBRC 3288 with those of the cellulose-producing strains clarified the genes important for cellulose production in Gluconacetobacter.

Complete Genome Sequence of Finegoldia magna, an Anaerobic Opportunistic Pathogen

Finegoldia magna (formerly Peptostreptococcus magnus), a member of the Gram-positive anaerobic cocci (GPAC), is a commensal bacterium colonizing human skin and mucous membranes. Moreover, it is also recognized as an opportunistic pathogen responsible for various infectious diseases. Here, we report the complete genome sequence of F. magna ATCC 29328. The genome consists of a 1 797 577 bp circular chromosome and an 189 163 bp plasmid (pPEP1). The metabolic maps constructed based on the genome information confirmed that most F. magna strains cannot ferment most sugars, except fructose, and have various aminopeptidase activities. Three homologs of albumin-binding protein, a known virulence factor useful for antiphagocytosis, are encoded on the chromosome, and one albumin-binding protein homolog is encoded on the plasmid. A unique feature of the genome is that F. magna encodes many sortase genes, of which substrates may be involved in bacterial pathogenesis, such as antiphagocytosis and adherence to the host cell. The plasmid pPEP1 encodes seven sortase and seven substrate genes, whereas the chromosome encodes four sortase and 19 substrate genes. These plasmid-encoded sortases may play important roles in the pathogenesis of F. magna by enriching the variety of cell wall anchored surface proteins.

Genome-wide phylogenetic analysis of Gluconobacter, Acetobacter, and Gluconacetobacter

Phylogenetic relationships among three genera, Gluconobacter, Acetobacter, and Gluconacetobacter, of acetic acid bacteria (AAB) are still unclear, although phylogenetic analysis using 16S rRNA gene sequence has shown that Gluconacetobacter diverged first from the ancestor of these three genera. Therefore, the relationships among these three genera were investigated by genome-wide phylogenetic analysis of AAB. Contrary to the results of 16S rRNA gene analysis, phylogenetic analysis of 293 enzymes involved in metabolism clearly showed that Gluconobacter separated first from its common ancestor with Acetobacter and Gluconacetobacter. In addition, we defined 753 unique orthologous proteins among five known complete genomes of AAB, and phylogenetic analysis was carried out using concatenated gene sequences of these 753 proteins. The result also showed that Gluconobacter separated first from its common ancestor with Acetobacter and Gluconacetobacter. Our results strongly suggest that Gluconobacter was the first to diverge from the common ancestor of Gluconobacter, Acetobacter, and Gluconacetobacter, a relationship that is in good agreement with the physiologies and habitats of these genera.

Adaptive mutation of Acetobacter pasteurianus SKU1108 enhances acetic acid fermentation ability at high temperature.

In vitro adaptation is one of the most challenging subjects in biology to understand adaptive evolution. Microbial adaptation to temperature is not only interesting in terms of understanding the adaptation mechanism, but also useful for industrial applications. In this study, we attempted the in vitro adaptation of Acetobacter pasteurianus SKU1108 by repeating its cultivation under high-temperature acetic acid fermentation conditions. As a result, thermo-adapted strains having the higher fermentation ability than the wild-type strain were obtained. Mutations and/or disruptions in several proteins of the adapted strains were detected with NGS sequencing technology. In particular, two different adapted strains had mutations or disruptions in three specific genes in common, suggesting that these genes are essential for thermotolerance or fermentation at higher temperature. In order to clarify their involvement in thermotolerance, two of the three genes were disrupted and their phenotype was examined. The results showed that mutations of the two proteins, MarR and an amino acid transporter, are partly responsible for higher fermentation ability and/or thermotolerance. Thus, it was suggested that these elevated abilities of the adapted strains are acquired by assembling several single gene mutations including the above two mutations.

Cyanide-insensitive Quinol Oxidase (CIO) from Gluconobacter oxydans is a unique terminal oxidase subfamily of cytochrome bd

Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5-6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.

Global analysis of the genes involved in the thermotolerance mechanism of thermotolerant Acetobacter tropicalis SKU1100.

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.

Genome-wide phylogenetic analysis of differences in thermotolerance among closely related Acetobacter pasteurianus strains.

Acetobacter pasteurianus is a Gram-negative strictly aerobic bacterium that is widely used for the industrial production of vinegar. Three Acetobacter pasteurianus strains, SKU1108, NBRC 3283 and IFO 3191, have the same 16S rRNA sequence (100 % sequence identity) but show differences in thermotolerance. To clarify the relationships between phylogeny and thermotolerance of these strains, genome-wide analysis of these three strains was performed. Concatenated phylogenetic analysis of a dataset of 1864 orthologues has shown that the more thermotolerant strains, SKU1108 and NBRC 3283, are more closely related to each other than to the more thermosensitive strain, IFO 3191. In addition, we defined a dataset of 2010 unique orthologues among these three strains, and compared the frequency of amino acid mutations among them. Genes involved in translation, transcription and signal transduction are highly conserved among each unique orthologous dataset. The results also showed that there are several genes with increased mutation rates in IFO 3191 compared with the thermotolerant strains, SKU1108 and NBRC 3283. Analysis of the mutational directions of these genes suggested that some of them might be correlated with the thermosensitivity of IFO 3191. Concatenated phylogenetic analysis of these closely related strains revealed that there is a phylogenetic relationship associated with this phenotype among the thermotolerant and thermosensitive strains.

Diagnostic Assay for Rickettsia japonica

We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project. The target ORF was present only in R. japonica–related strains.