Yoshinao Azuma

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.

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.

Phosphoinositide 3-kinase in nitric oxide synthesis in macrophage : Critical dimerization of inducible nitric-oxide synthase

Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85α regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-γ stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.

RhoH Plays Critical Roles in FcεRI-Dependent Signal Transduction in Mast Cells

RhoH is an atypical small G protein with defective GTPase activity that is specifically expressed in hematopoietic lineage cells. RhoH has been implicated in regulation of several physiological processes including hematopoiesis, integrin activation, and T cell differentiation and activation. In the present study, we investigated the role of RhoH in mast cells by generating RhoH knockout mice. Despite observing normal development of mast cells in vivo, passive systemic anaphylaxis and histamine release were impaired in these mice. We also observed defective degranulation and cytokine production upon FcεRI ligation in RhoH-deficient bone marrow-derived mast cells. Furthermore, FcεRI-dependent activation of Syk and phosphorylation of its downstream targets, including LAT, SLP76, PLCγ1, and PLCγ2 were impaired, however phosphorylation of the γ-subunit of FcεRI remained intact. We also found RhoH-Syk association that was greatly enhanced by active Fyn. Our results indicate that RhoH regulates FcεRI signaling in mast cells by facilitating Syk activation, possibly as an adaptor molecule for Syk.

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.

Rac1-mediated Bcl-2 induction is critical in antigen-induced CD4 single-positive differentiation of a CD4+CD8+ immature thymocyte line

Rac1, one of the Rho family small guanosine triphosphatases, has been shown to work as a “molecular switch” in various signal transduction pathways. To assess the function of Rac1 in the differentiation process of CD4 single‐positive (CD4‐SP) T cells from CD4CD8 double‐positive (DP) cells, we used a DP cell line DPK, which can differentiate into CD4‐SP cells upon TCR stimulation in vitro. DPK expressing dominant‐negative (dn)Rac1 underwent massive apoptosis upon TCR stimulation and resulted in defective differentiation of CD4‐SP cells. Conversely, overexpression of dnRac2 did not affect differentiation. TCR‐dependent actin polymerization was inhibited, whereas early ERK activation was unaltered in dnRac1‐expressing DPK. We found that TCR‐dependent induction of Bcl‐2 was suppressed greatly in dnRac1‐expressing DPK, and this suppression was independent of actin rearrangement. Furthermore, introduction of exogenous Bcl‐2 inhibited TCR‐dependent induction of apoptosis and restored CD4‐SP generation in dnRac1‐expressing DPK without restoring TCR‐induced actin polymerization. Collectively, these data indicate that Rac1 is critical in differentiation of CD4‐SP from the DP cell line by preventing TCR‐induced apoptosis via Bcl‐2 up‐regulation.

Adaptive mutation related to cellulose producibility in Komagataeibacter medellinensis (Gluconacetobacter xylinus) NBRC 3288

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.

Genomic analyses of thermotolerant microorganisms used for high-temperature fermentations.

Environmental adaptation is considered as one of the most challenging subjects in biology to understand evolutionary or ecological diversification processes and in biotechnology to obtain useful microbial strains. Temperature is one of the important environmental stresses; however, microbial adaptation to higher temperatures has not been studied extensively. For industrial purposes, the use of thermally adapted strains is important, not only to reduce the cooling expenses of the fermentation system, but also to protect fermentation production from accidental failure of thermal management. Recent progress in next-generation sequencing provides a powerful tool to track the genomic changes of the adapted strains and allows us to compare genomic DNA sequences of conventional strains with those of their closely related thermotolerant strains. In this article, we have attempted to summarize our recent approaches to produce thermotolerant strains by thermal adaptation and comparative genomic analyses of Acetobacter pasteurianus for high-temperature acetic acid fermentations, and Zymomonas mobilis and Kluyveromyces marxianus for high-temperature ethanol fermentations. Genomic analysis of the adapted strains has found a large number of mutations and/or disruptions in highly diversified genes, which could be categorized into groups related to cell surface functions, ion or amino acid transporters, and some transcriptional factors. Furthermore, several phenotypic and genetic analyses revealed that the thermal adaptation could lead to decreased ROS generation in cells that produce higher ROS levels at higher temperatures. Thus, it is suggested that the thermally adapted cells could become robust and resistant to many stressors, and thus could be useful for high-temperature fermentations. Graphical abstract Experimental (in vitro) adaptation, as well as natural adaptation, could generate thermotolerant species, useful for high-temperature fermentation, from mesophiles.

Complete genome and gene expression analyses of Asaia bogorensis reveal unique responses to culture with mammalian cells as a potential opportunistic human pathogen

Asaia bogorensis, a member of acetic acid bacteria (AAB), is an aerobic bacterium isolated from flowers and fruits, as well as an opportunistic pathogen that causes human peritonitis and bacteraemia. Here, we determined the complete genomic sequence of the As. bogorensis type strain NBRC 16594, and conducted comparative analyses of gene expression under different conditions of co-culture with mammalian cells and standard AAB culture. The genome of As. bogorensis contained 2,758 protein-coding genes within a circular chromosome of 3,198,265 bp. There were two complete operons encoding cytochrome bo3-type ubiquinol terminal oxidases: cyoABCD-1 and cyoABCD-2. The cyoABCD-1 operon was phylogenetically common to AAB genomes, whereas the cyoABCD-2 operon belonged to a lineage distinctive from the cyoABCD-1 operon. Interestingly, cyoABCD-1 was less expressed under co-culture conditions than under the AAB culture conditions, whereas the converse was true for cyoABCD-2. Asaia bogorensis shared pathogenesis-related genes with another pathogenic AAB, Granulibacter bethesdensis, including a gene coding pathogen-specific large bacterial adhesin and additional genes for the inhibition of oxidation and antibiotic resistance. Expression alteration of the respiratory chain and unique hypothetical genes may be key traits that enable the bacterium to survive under the co-culture conditions.

An epistatic effect of apaf-1 and caspase-9 on chlamydial infection

Chlamydia is an obligate intracellular bacterial pathogen that replicates solely within a membrane-bound vacuole termed an inclusion. Chlamydia seems to perturb multiple cellular processes of the host, such as, rearrangement of the membrane trafficking system for its intracellular multiplication, and inhibition of host cell apoptosis for persistent infection. In an attempt to clarify host factor involvement in apoptosis regulation, we found that inhibition of Caspase-9 restricted, while Apaf-1 promoted, Chlamydia pneumoniae infection in HEp-2, HeLa, and mouse epithelial fibroblast (MEF) cells. These opposition contributions to the chlamydial infection were confirmed using caspase-9−/− and apaf-1−/− MEFs. Similar phenomena also appeared in the case of infection with Chlamydia trachomatis. Interestingly, caspase-9 in apaf-1−/− MEFs was activated by chlamydial infection but during the infection caspase-3 was not activated. That is, caspase-9 was activated without support for multiplication and activation by Apaf-1, and the activated caspase-9 may be physically disconnected from the caspase cascade. This may be partially explained by the observation of caspase-9 accumulation within chlamydial inclusions. The sequestration of caspase-9 by chlamydia seems to result in apoptosis repression, which is crucial for the chlamydial development cycle. Because Apaf-1 shares domains with intracellular innate immune receptor NOD1, it may play a key role in the strategy to regulate chlamydial infection.