Effect of estradiol regulating sortilin-related receptor A expression on hippocampal spine density and synaptic protein expression via estrogen receptor of mouse / 解剖学报

Objective To study the effect and mechanism of estradiol (E

Characterization of a Protease Hyper-Productive Mutant of Bacillus pumilus by Comparative Genomic and Transcriptomic Analysis.

Bacillus pumilus BA06 has great potential for the production of alkaline proteases. To improve the protease yield, classical mutagenesis to combine the physical and chemical mutagens was performed to obtain a protease hyper-productive mutant SCU11. The full genome sequences of BA06 and SCU11 strains were assembled through DNA sequencing using the PacBio sequencing platform. By comparative genomics analysis, 147 SNPs and 15 InDels were found between these two genomes, which lead to alternation of coding sequence in 15 genes. Noticeable, the gene (kinA) encoding sporulation kinase A is interrupted by introducing a stop codon in its coding region in BA06. Interestedly, this gene is reversely corrected in SCU11. Furthermore, comparative transcriptome analysis revealed that kinA and two positive regulatory genes (DegU and Spo0A) were upregulated in transcription in SCU11. In terms of the transcriptional data, upregulation of a phosphorylation cascade starting with KinA may enhance Spo0A phosphorylation, and thus activate expression of the gene aprE (encoding major extracellular protease) through repression of AbrB (a repressor of aprE) and activation of SinI, an antagonist of SinR (a repressor of aprE). In addition, the other genes involved in various metabolic pathways, especially of membrane transport and sporulation, were altered in transcription between these two strains. Conclusively, our transcriptome data suggested that upregulation degU and spo0A, as well as kinA, may at least partially contribute to the high production of alkaline protease in SCU11.

Construction of a High-Expression System in Bacillus through Transcriptomic Profiling and Promoter Engineering.

Bacillus subtilis is an ideal host for secretion and expression of foreign proteins. The promoter is one of the most important elements to facilitate the high-level production of recombinant protein. To expand the repertoire of strong promoters for biotechnological applications in Bacillus species, 14 highly transcribed genes based on transcriptome profiling of B. pumilus BA06 were selected and evaluated for their promoter strength in B. subtilis. Consequently, a strong promoter P2069 was obtained, which could drive the genes encoding alkaline protease (aprE) and green fluorescent protein (GFP) to express more efficiency by an increase of 3.65-fold and 18.40-fold in comparison with the control promoter (PaprE), respectively. Further, promoter engineering was applied to P2069, leading to a mutation promoter (P2069M) that could increase GFP expression by 3.67-fold over the wild-type promoter (P2069). Moreover, the IPTG-inducible expression systems were constructed using the lac operon based on the strong promoters of P2069 and P2069M, which could work well both in B. subtilis and B. pumilus. In this study, highly efficient expression system for Bacillus was constructed based on transcriptome data and promoter engineering, which provide not only a new option for recombinant expression in B. subtilis, but also novel genetic tool for B. pumilus.

A novel DNA methylation motif identified in Bacillus pumilus BA06 and possible roles in the regulation of gene expression.

Single-molecule real-time (SMRT) sequencing can be used to identify a wide variety of chemical modifications of the genome, such as methylation. Here, we applied this approach to identify N6-methyl-adenine (m6A) and N4-methyl-cytosine (m4C) modification in the genome of Bacillus pumilus BA06. A typical methylation recognition motif of the type I restriction-modification system (R-M), 5'-TCm6AN8TTGG-3'/3'-AGTN8m6AACC-5', was identified. We confirmed that this motif was a new type I methylation site using REBASE analysis and that it was recognized by a type I R-M system, Bpu6ORFCP, according to methylation sensitivity assays in vivo and vitro. Furthermore, we found that deletion of the R-M system Bpu6ORFCP induced transcriptional changes in many genes and led to increased gene expression in pathways related to ABC transporters, sulfur metabolism, ribosomes, cysteine and methionine metabolism and starch and sucrose metabolism, suggesting that the R-M system in B. pumilus BA06 has other significant biological functions beyond protecting the B. pumilus BA06 genome from foreign DNA.

Disruption of the pleiotropic gene scoC causes transcriptomic and phenotypical changes in Bacillus pumilus BA06.

BACKGROUND: Bacillus pumilus is a Gram-positive and endospore-forming bacterium broadly existing in a variety of environmental niches. Because it produces and secrets many industrially useful enzymes, a lot of studies have been done to understand the underlying mechanisms. Among them, scoC was originally identified as a pleiotropic transcription factor negatively regulating protease production and sporulation in B. subtilis. Nevertheless, its role in B. pumilus largely remains unknown. RESULTS: In this study we successfully disrupted scoC gene in B. pumilus BA06 and found increased total extracellular protease activity in scoC mutant strain. Surprisingly, we also found that scoC disruption reduced cell motility possibly by affecting flagella formation. To better understand the underlying mechanism, we performed transcriptome analysis with RNA sequencing. The result showed that more than one thousand genes were alternated at transcriptional level across multiple growth phases, and among them the largest number of differentially expressed genes (DEGs) were identified at the transition time point (12 h) between the exponential growth and the stationary growth phases. In accordance with the altered phenotype, many protease genes especially the aprE gene encoding alkaline protease were transcriptionally regulated. In contrast to the finding in B. subtilis, the aprN gene encoding neutral protease was transcriptionally downregulated in B. pumilus, implicating that scoC plays strain-specific roles. CONCLUSIONS: The pleiotropic transcription factor ScoC plays multiple roles in various cellular processes in B. pumilus, some of which were previously reported in B. subtilis. The supervising finding is the identification of ScoC as a positive regulator for flagella formation and bacterial motility. Our transcriptome data may provide hints to understand the underlying mechanism.

Transcriptome profiling analysis reveals metabolic changes across various growth phases in Bacillus pumilus BA06.

BACKGROUND: Bacillus pumilus can secret abundant extracellular enzymes, and may be used as a potential host for the industrial production of enzymes. It is necessary to understand the metabolic processes during cellular growth. Here, an RNA-seq based transcriptome analysis was applied to examine B. pumilus BA06 across various growth stages to reveal metabolic changes under two conditions. RESULTS: Based on the gene expression levels, changes to metabolism pathways that were specific to various growth phases were enriched by KEGG analysis. Upon entry into the transition from the exponential growth phase, striking changes were revealed that included down-regulation of the tricarboxylic acid cycle, oxidative phosphorylation, flagellar assembly, and chemotaxis signaling. In contrast, the expression of stress-responding genes was induced when entering the transition phase, suggesting that the cell may suffer from stress during this growth stage. As expected, up-regulation of sporulation-related genes was continuous during the stationary growth phase, which was consistent with the observed sporulation. However, the expression pattern of the various extracellular proteases was different, suggesting that the regulatory mechanism may be distinct for various proteases. In addition, two protein secretion pathways were enriched with genes responsive to the observed protein secretion in B. pumilus. However, the expression of some genes that encode sporulation-related proteins and extracellular proteases was delayed by the addition of gelatin to the minimal medium. CONCLUSIONS: The transcriptome data depict global alterations in the genome-wide transcriptome across the various growth phases, which will enable an understanding of the physiology and phenotype of B. pumilus through gene expression.