Antisense RNA associated with biological regulation of a restriction-modification system.

Restriction-modification systems consist of a modification enzyme that methylates a specific DNA sequence and a restriction endonuclease that cleaves DNA lacking this epigenetic signature. Their gene expression should be finely regulated because their potential to attack the host bacterial genome needs to be controlled. In the EcoRI system, where the restriction gene is located upstream of the modification gene in the same orientation, we previously identified intragenic reverse promoters affecting gene expression. In the present work, we identified a small (88 nt) antisense RNA (Rna0) transcribed from a reverse promoter (P(REV0)) at the 3' end of the restriction gene. Its antisense transcription, as measured by transcriptional gene fusion, appeared to be terminated by the P(M1,M2) promoter. P(M1,M2) promoter-initiated transcription, in turn, appeared to be inhibited by P(REV0). Mutational inactivation of P(REV0) increased expression of the restriction gene. The biological significance of this antisense transcription is 2-fold. First, a mutation in P(REV0) increased restriction of incoming DNA. Second, the presence of the antisense RNA gene (ecoRIA) in trans alleviated cell killing after loss of the EcoRI plasmid (post-segregational killing). Taken together, these results strongly suggested the involvement of an antisense RNA in the biological regulation of this restriction-modification system.

Nitrite accumulation, denitrification kinetic and microbial evolution in the partial denitrification process: The combined effects of carbon source and nitrate concentration.

The combined effects of carbon source (HAc, HPr, Glu, Glu + HAc) and nitrate concentration (40, 80 mg/L labeling as R40, R80) on partial denitrification (PD) were discussed at C/N ratio of 2.5 (COD = 100, 200 mg/L). The optimal NO2--N and NTR reached to 67.03 mg/L, 99.14% in HAc-R80 system, and denitrification kinetics revealed the same conclusion, corresponding to higher COD utilization rate (CUR: 58.46 mgCOD/(gVSS·h)), nitrate reduction rate (NaRR: 29.94 mgN/(gVSS·h)) and nitrite accumulation rate (NiAR: 29.68 mgN/(gVSS·h)). The preference order was HAc > HPr > Glu + HAc > Glu in both R40 and R80 systems due to different metabolic pathways, however, the NO2--N accumulation and kinetic parameters of R80 group were dramatically higher than those in R40 for the same carbon source. The R80 group facilitated more concentrated biodiversity (607-808 OTUs) with Terrimonas and norank_f_Saprospiraceae responsible for high NO2--N accumulation in HAc and HPr served systems, while norank_f_norank_o_Saccharimonadales and OLB13 dominated the Glu containing systems.

Comparisons of nitrite accumulation, microbial behavior and nitrification kinetic in continuous stirred tank (ST) and plug flow (PF) moving bed biofilm reactors.

Two types of continuous stirred tank moving bed biofilm reactors (ST-MBBR) and plug flow MBBR (PF-MBBR) were compared for nitrification. PF-MBBR showed strong shock resistance to temperature, and ammonium oxidation ratio (AOR) was 9.63% higher than that in the ST-MBBR, although the average biomass and biofilm thickness of ST-MBBR were 7.32-18.59%, 9.44-14.06% higher than those in the PF-MBBR. Meanwhile, a lower nitrite accumulation ratio (NAR) was observed (54.88%) in the PF-MBBR than the ST-MBBR (78.92%) due to different operation modes, and the divergence was demonstrated by the microbial quantitative analysis. Nitrification kinetics revealed that the temperature coefficient (θ) in the ST-MBBR (1.068) was much higher than that in the PF-MBBR (1.006-1.015), proving the contrasting nitrification performances caused by temperature shock. According to the Monod equation, the half-saturation coefficient (KN) in the ST-MBBR was 0.19 mg/L while it varied around 0.12-0.24 mg/L in the PF-MBBR, revealing various NH4+ affinity owing to different biofilm thickness and microbial composition. Finally, MBBR optimization related to operation mode, temperature, and free ammonium (FA) inhibition for nitrite accumulation was discussed.

The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome.

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant DeltaN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in DeltaN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.