Although n-butanol (BuOH) is an ideal fuel because of its superior physical properties, it has toxicity to microbes. Previously, a Synechococcus elongatus PCC 7942 derivative strain that produces BuOH from CO₂ was developed by introducing six heterologous genes (BUOH-SE strain). To identify the bottleneck in BuOH production, the effects of BuOH production and its toxicity on central metabolism and the photosystem were investigated. Parental (WT) and BUOH-SE strains were cultured under autotrophic conditions. Consistent with the results of a previous study, BuOH production was observed only in the BUOH-SE strain. Isotopically non-stationary ¹³C-metabolic flux analysis revealed that the CO₂ fixation rate was much larger than the BuOH production rate in the BUOH-SE strain (1.70 vs 0.03 mmol gDCW^-1 h^-1), implying that the carbon flow for BuOH biosynthesis was less affected by the entire flux distribution. No large difference was observed in the flux of metabolism between the WT and BUOH-SE strains. Contrastingly, in the photosystem, the chlorophyll content and maximum O₂ evolution rate per dry cell weight of the BUOH-SE strain were decreased to 81% and 43% of the WT strain, respectively. Target proteome analysis revealed that the amounts of some proteins related to antennae (ApcA, ApcD, ApcE, and CpcC), photosystem II (PsbB, PsbU, and Psb28-2), and cytochrome b₆f complex (PetB and PetC) in photosystems decreased in the BUOH-SE strain. The activation of photosynthesis would be a novel approach for further enhancing BuOH production in S. elongatus PCC 7942.

The Escherichia coli genome was searched for potential terminators of the rolling-circle replication of staphylococcal plasmid pC194. The replication origin of pC194 was randomly inserted into the E. coli chromosome and rolling-circle replication was initiated by producing pC194’s replication protein from a plasmid. Circular DNA resulting from termination in the chromosome was recovered from 42 of the 100 insertion clones screened. The nucleotide sequences at the ends of the chromosomal segment in the recovered DNA were determined and used to identify the locus of integration and the point of termination. The sequence beyond the termination point was retrieved from the database. This information would have been unrecoverable if synthetic random sequences had been used for screening. The consensus sequence based on the discovered potential terminators was consistent with the results of previous and new experiments. The recovered circular DNAs contain a hybrid origin consisting of a 5’ part derived from the chromosomal DNA and a 3’ part of the integrated origin. Two such hybrid origins were examined for initiation function and shown to be as effective as the authentic pC194 origin. These results suggest a possible evolutionary mechanism in which a rolling-circle plasmid may acquire genes from the host organism.

α-1,3-Glucanase Agl-KA from Bacillus circulans KA-304 consists of a discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), a threonine-proline-rich-linker (TP linker), a discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. The binding of DS1, CBM6, and DS2 to α-1,3-glucan can be improved in the presence of two of these three domains. In this study, DS1, CBM6, and TP linker were genetically fused to histamine dehydrogenase (HmDH) from Nocardioides simplex NBRC 12069. The fusion enzyme, AGBDs-HmDH, was expressed in Escherichia coli Rosetta 2 (DE3) and purified from the cell-free extract. AGBDs-HmDH bound to 1% micro-particle of α-1,3-glucan (diameter: less than 1 μm) and 7.5% coarse-particle of α-1,3-glucan (less than 200 μm) at about 97 % and 70% of the initial amounts of the enzyme, respectively. A reactor for flow injection analysis filled with AGBDs-HmDH immobilized on the coarse-particle of α-1,3-glucan was successfully applied to determine histamine. A linear calibration curve was observed in the range for about 0.1 to 3.0 mM histamine. These findings suggest that the combination of α-1,3-glucan and α-1,3-glucan binding domains is a candidate for novel enzyme immobilization.

When Bacillus subtilis cells face environmental deterioration, such as exhaustion of nutrients and an increase in cell density, they form spores. It is known that phosphorylation of Spo0A and activation of σ^H are key events at the initiation of sporulation. However, the initiation of sporulation is an extremely complicated process, and the relationship between these two events remains to be elucidated. To determine the minimum requirements for triggering sporulation initiation, we attempted to induce cell sporulation at the log phase, regardless of nutrients and cell density. In rich media such as Luria-Bertani (LB) medium, the cells of B. subtilis do not sporulate efficiently, possibly because of excess nutrition. When the amount of xylose in the LB medium was limited, σ^H -dependent transcription of the strain, in which sigA was under the control of the xylose-inducible promoter, was induced, and the frequency of sporulation was elevated according to the decreased level of σ^A. We also employed a fusion of sad67, which codes for an active form of Spo0A, and the IPTG-inducible promoter. The combination of lowered σ^A expression and activated Spo0A allowed the cells in the log phase to stop growing and rush into spore development. This observation of enforced initiation of sporulation in the mutant strain was detected even in the presence of the wild-type strain, suggesting that only intracellular events initiate and fulfill spore development regardless of extracellular conditions. Under natural sporulation conditions, the amount of σ^A did not change drastically throughout growth. Mechanisms that sequester σ^A from the core RNA polymerase and help σ^H to become active exist, but this has not yet been elucidated.

Arginine is a proteinogenic amino acid that organisms additionally exploit both for nitrogen storage and as a stress protectant. The location of arginine, whether intra- or extracellular, is important in maintaining physiological homeostasis. Here, we identified an arginine transporter ortholog of the emerging fungal pathogenic Candida glabrata. Blast searches revealed that the C. glabrata genome contains two potential orthologs of the Saccharomyces cerevisiae arginine transporter gene CAN1 (CAGL0J08162g and CAGL0J08184g). We then found that CAGL0J08162g is stably located on the plasma membrane and performs cellular uptake of arginine. Moreover, CAGL0J08162-disrupted cells of C. glabrata showed a partial resistance to canavanine, a toxic analog of arginine. Our data suggest that CAGL0J08162g is a key arginine transporter in the pathogenic C. glabrata (CgCan1).

Six aromatic secondary metabolites, pestalone (1), emodin (2), phomopsilactone (3), pestalachlorides B (4), C (5), and D (6), were isolated from Pestalotiopsis sp. FKR-0115, a filamentous fungus collected from white moulds growing on dead branches in Minami Daito Island. The efficacy of these secondary metabolites against methicillin-resistant Staphylococcus aureus (MRSA) with and without meropenem (β-lactam antibiotic) was evaluated using the paper disc method and broth microdilution method. The chemical structures of the isolated compounds (1–6) were characterised using spectroscopic methods, including nuclear magnetic resonance and mass spectrometry. All six isolated compounds exhibited synergistic activity with meropenem against MRSA. Among the six secondary metabolites, pestalone (1) overcame bacterial resistance in MRSA to the greatest extent.