Usefulness of F-18 FDG PET/CT in a Case of Relapsing Polychondritis

Article信州医学雑誌 64(6): 349-355(2016)journal articl

Enhanced Recombinant Protein Productivity by Genome Reduction in Bacillus subtilis

The emerging field of synthetic genomics is expected to facilitate the generation of microorganisms with the potential to achieve a sustainable society. One approach towards this goal is the reduction of microbial genomes by rationally designed deletions to create simplified cells with predictable behavior that act as a platform to build in various genetic systems for specific purposes. We report a novel Bacillus subtilis strain, MBG874, depleted of 874 kb (20%) of the genomic sequence. When compared with wild-type cells, the regulatory network of gene expression of the mutant strain is reorganized after entry into the transition state due to the synergistic effect of multiple deletions, and productivity of extracellular cellulase and protease from transformed plasmids harboring the corresponding genes is remarkably enhanced. To our knowledge, this is the first report demonstrating that genome reduction actually contributes to the creation of bacterial cells with a practical application in industry. Further systematic analysis of changes in the transcriptional regulatory network of MGB874 cells in relation to protein productivity should facilitate the generation of improved B. subtilis cells as hosts of industrial protein production

Cell Size and the Initiation of DNA Replication in Bacteria

In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ∼30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation. Prompted by this finding, we re-examined the link between cell size and initiation in both E. coli and B. subtilis. Although changes in DNA replication have been shown to alter both E. coli and B. subtilis cell size, the converse (the effect of cell size on DNA replication) has not been explored. Here, we report that the mechanisms responsible for coordinating DNA replication with cell size vary between these two model organisms. In contrast to B. subtilis, small E. coli mutants delayed replication initiation until they achieved the size at which wild-type cells initiate. Modest increases in DnaA alleviated the delay, supporting the view that growth-dependent accumulation of DnaA is the trigger for replication initiation in E. coli. Significantly, although small E. coli and B. subtilis cells both maintained wild-type concentration of DnaA, only the E. coli mutants failed to initiate on time. Thus, rather than the concentration, the total amount of DnaA appears to be more important for initiation timing in E. coli. The difference in behavior of the two bacteria appears to lie in the mechanisms that control the activity of DnaA

Indirect positive effects of a sigma factor RpoN deletion on the lactate-based polymer production in Escherichia coli

The production of bacterial polyesters, polyhydroxyalkanoates (PHAs), has been improved by several rational approaches such as overexpression and/or engineering of the enzymes directly related to PHA biosynthetic pathways. In this study, a new approach at transcription level has been applied to a new category of the copolymer of lactate (LA) and 3-hydroxybutyrate (3HB), P(LA-co-3HB). When the 4 disrupting mutants of sigma factors in Escherichia coli, rpoN, rpoS, fliA, fecI, were used as platforms for production of P(LA-co-3HB), increases in the production level and LA fraction of the copolymer were observed for the mutant strain with rpoN disruption. These positive impacts on the polymer production were caused in an indirect manner via changes in the multiple genes governed by RpoN. A genome-wide engineering by sigma factors would be a versatile approach for the production of value-added products of interest and available for combination with the other beneficial tools

Energy and water management A guide for schools

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Molecular weight-dependent degradation of D-lactate-containing polyesters by polyhydroxyalkanoate depolymerases from Variovorax sp C34 and Alcaligenes faecalis T1

Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZ(Vs)) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(d-LA-co-d-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZ(Vs) for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZ(Af)) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZ(Vs) generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZ(Vs) cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZ(Vs) but also PhaZ(Af) hydrolyzed all of these substrates, namely PhaZ(Af) also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZ(Vs) exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZ(Af). Therefore, the cleaving capability of PhaZs used here toward the d-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme