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BackgroundMost bacteria can use various compounds as carbon sources. These carbon sources can be either co-metabolized or sequentially metabolized, where the latter phenomenon typically occurs as catabolite repression. From the practical application point of view of utilizing lignocellulose for the production of biofuels etc., it is strongly desirable to ferment all sugars obtained by hydrolysis from lignocellulosic materials, where simultaneous consumption of sugars would benefit the formation of bioproducts. However, most organisms consume glucose prior to consumption of other carbon sources, and exhibit diauxic growth. It has been shown by fermentation experiments that simultaneous consumption of sugars can be attained by ptsG, mgsA mutants etc., but its mechanism has not been well understood. It is strongly desirable to understand the mechanism of metabolic regulation for catabolite regulation to improve the performance of fermentation.ResultsIn order to make clear the catabolic regulation mechanism, several continuous cultures were conducted at different dilution rates of 0.2, 0.4, 0.6 and 0.7 h-1 using wild type Escherichia coli. The result indicates that the transcript levels of global regulators such as crp, cra, mlc and rpoS decreased, while those of fadR, iclR, soxR/S increased as the dilution rate increased. These affected the metabolic pathway genes, which in turn affected fermentation result where the specific glucose uptake rate, the specific acetate formation rate, and the specific CO2 evolution rate (CER) were increased as the dilution rate was increased. This was confirmed by the 13C-flux analysis. In order to make clear the catabolite regulation, the effect of crp gene knockout (Δcrp) and crp enhancement (crp+) as well as mlc, mgsA, pgi and ptsG gene knockout on the metabolism was then investigated by the continuous culture at the dilution rate of 0.2 h-1 and by some batch cultures. In the case of Δcrp (and also Δmlc) mutant, TCA cycle and glyoxylate were repressed, which caused acetate accumulation. In the case of crp+ mutant, glycolysis, TCA cycle, and gluconeogenesis were activated, and simultaneous consumption of multiple carbon sources can be attained, but the glucose consumption rate became less due to repression of ptsG and ptsH by the activation of Mlc. Simultaneous consumption of multiple carbon sources could be attained by mgsA, pgi, and ptsG mutants due to increase in crp as well as cyaA, while glucose consumption rate became lower.ConclusionsThe transcriptional catabolite regulation mechanism was made clear for the wild type E. coli, and its crp, mlc, ptsG, pgi, and mgsA gene knockout mutants. The results indicate that catabolite repression can be relaxed and crp as well as cyaA can be increased by crp+, mgsA, pgi, and ptsG mutants, and thus simultaneous consumption of multiple carbon sources including glucose can be made, whereas the glucose uptake rate became lower as compared to wild type due to inactivation of ptsG in all the mutants considered.

Pyrrole−imidazole polyamides (PIPs) bind to predetermined double-stranded DNA sequences and selectively target a large variety of DNA sequences. Although the forward-binding (5′-3′/N−C) orientation, in which the N-terminus of PIPs faces the 5′-terminus of DNAs, is considered to be the main binding manner of PIPs, a reverse-binding (5′-3′/C−N) orientation, in which the C-terminus of PIPs faces the 3′-terminus of DNAs, sometimes causes unintended binding. Here, we synthesized optical or structural isomers of previously reported cyclic PIPs (cPIPs), which differ in the position of the amino groups in the γ-turn units, and we investigated their binding affinities both in the forward-and reverse-binding orientation. We show that cPIPs with (R)-α-amino-γ-turn units prefer the forward orientation as do hairpin PIPs. More importantly, we document for the first time the remarkable reversebinding preference of cPIPs with (S)-α-amino-γ-turns. These results indicate that the orientation preference of cPIPs can be controlled by the position of the amino groups on the γ-turn units, which may markedly increase the number of DNA sequences that can be targeted by PIPs.

Consecutive guanine RNA sequences can adopt quadruple-stranded structures, termed RNA G-quadruplexes (rG4s). Although rG4-forming sequences are abundant in transcriptomes, the physiological roles of rG4s in the central nervous system remain poorly understood. In the present study, proteomics analysis of the mouse forebrain identified DNAPTP6 as an RNA binding protein with high affinity and selectivity for rG4s. We found that DNAPTP6 coordinates the assembly of stress granules (SGs), cellular phase-separated compartments, in an rG4-dependent manner. In neurons, the knockdown of DNAPTP6 diminishes the SG formation under oxidative stress, leading to synaptic dysfunction and neuronal cell death. rG4s recruit their mRNAs into SGs through DNAPTP6, promoting RNA self-assembly and DNAPTP6 phase separation. Together, we propose that the rG4-dependent phase separation of DNAPTP6 plays a critical role in neuronal function through SG assembly.

Abnormally expanded CAG/CTG repeat DNA sequences lead to a variety of neurological diseases, such as Huntington's disease.Here, we synthesized a cyclic pyrrole-imidazole polyamide (cPIP), which can bind to the minor groove of the CAG/CTG DNA sequence. The double-stranded DNA melting temperature (T m ) and surface plasmon resonance assays revealed the high binding affinity of the cPIP. In addition, next-generation sequencing showed that the cPIP had high specificity for its target DNA sequence.Although DNA repeat sequences are normally present in the human genome, abnormally elongated repeats can lead to a variety of diseases. [1,2] The abnormal elongation of CAG/CTG repeat sequences causes Huntington's disease, spinocerebellar ataxia, and myotonic dystrophy. Compounds that bind to the CAG/CTG repeat sequences have been studied to develop therapeutic agents for these neurological disorders. [3][4][5] Notably, Pearson et al. recently achieved a reduction in the number of repeats in vivo using a compound that binds to the hairpin structure formed by the CAG repeats. [6] These studies targeted the r(CUG) repeats [3,4] or d(CAG/CAG) hairpin structures in the CAG repeat regions, [5,6] whereas our group previously developed pyrrole-imidazole polyamides (PIPs) that bind to the d(CAG/ CTG) sequences in a sequence-specific manner. [7,8] PIPs are one of the well-studied DNA-binding compounds, which were developed by Dervan et al. [9,10] They bind to the minor groove of B-DNA, recognizing Watson-Crick base pairs by antiparallel pairings of their N-methylpyrrole (Py) and N-meth-[a] Y.