Expression, purification and characterization of CTP synthase PyrG in Staphylococcusaureus.

Staphylococcus aureus (S. aureus) presents a significant challenge in both nosocomial and community settings due to its pathogenicity. The emergence of drug-resistant strains exacerbates S. aureus infections, leading to increased mortality rates. PyrG, a member of the cytidine triphosphate (CTP) synthase family, serves as a crucial therapeutic target against S. aureus due to the pivotal role of CTP in cellular metabolism. However, the structural and mechanistic details of S. aureus PyrG remains unknown. Here, we successfully expressed and purified monomeric PyrG. Mutational experiments were conducted based on the results of molecular docking. Based on the results of the molecular docking, we carried out mutation experiments and found that Q386A dramatically decreased the CTP synthase activity compared to the wild-type protein, while Y54A almost completely abolished the activity. Exposure of S. aureus to the kinase inhibitor crizotinib increased expression of gene pyrG. Our results identify the two key sites on PyrG for the CTP synthase activity, and present PyrG gene expression increased during the treatment of crizotinib, which may eventually provide valuable guidance for the development of new drugs against S. aureus infections.

Integrated transcriptomic and proteomic analysis reveals the complex molecular mechanisms underlying stone cell formation in Korla pear.

Korla pear (Pyrus sinkiangensis Yü) is a landrace selected from a hybrid pear species in the Xinjiang Autonomous Region in China. In recent years, pericarp roughening has been one of the major factors that adversely affects fruit quality. Compared with regular fruits, rough-skin fruits have a greater stone cell content. Stone cells compose sclerenchyma tissue that is formed by secondary thickening of parenchyma cell walls. In this work, we determined the main components of stone cells by isolating them from the pulp of rough-skin fruits at the ripening stage. Stone cell staining and apoptosis detection were then performed on fruit samples that were collected at three different developmental stages (20, 50 and 80 days after flowering (DAF)) representing the prime, late and stationary stages of stone cell differentiation, respectively. The same batches of samples were used for parallel transcriptomic and proteomic analysis to identify candidate genes and proteins that are related to SCW biogenesis in Korla pear fruits. The results showed that stone cells are mainly composed of cellulose (52%), hemicellulose (23%), lignin (20%) and a small amount of polysaccharides (3%). The periods of stone cell differentiation and cell apoptosis were synchronous and primarily occurred from 0 to 50 DAF. The stone cell components increased abundantly at 20 DAF but then decreased gradually. A total of 24,268 differentially expressed genes (DEGs) and 1011 differentially accumulated proteins (DAPs) were identified from the transcriptomic and proteomic data, respectively. We screened the DEGs and DAPs that were enriched in SCW-related pathways, including those associated with lignin biosynthesis (94 DEGs and 31 DAPs), cellulose and xylan biosynthesis (46 DEGs and 18 DAPs), S-adenosylmethionine (SAM) metabolic processes (10 DEGs and 3 DAPs), apoplastic ROS production (16 DEGs and 2 DAPs), and cell death (14 DEGs and 6 DAPs). Among the identified DEGs and DAPs, 63 significantly changed at both the transcript and protein levels during the experimental periods. In addition, the majority of these identified genes and proteins were expressed the most at the prime stage of stone cell differentiation, but their levels gradually decreased at the later stages.

Characterization of two copper/zinc superoxide dismutases (Cu/Zn-SODs) from the desert beetle Microdera punctipennis and their activities in protecting E. coli cells against cold.

Superoxide dismutases (SODs) are crucial in scavenging reactive oxygen species (ROS); however, studies regarding SOD functions in insects under cold conditions are rare. In this paper, two novel Cu/Zn-SOD genes in the desert beetle Microdera punctipennis, an extracellular copper/zinc SOD (MpecCu/Zn-SOD) and an intracellular copper/zinc SOD (MpicCu/Zn-SOD), were identified and characterized. The results of quantitative real-time PCR showed that MpecCu/Zn-SOD expression was significantly up-regulated by 4 °C exposure for 0.5 h, but MpicCu/Zn-SOD was not. Superoxide anion radical (O2•-) content in beetles under 4 °C exposure for 0.5 h showed an initial sharp increase and fluctuated during the cold treatment period, which was consistent with the relative mRNA level of MpecCu/Zn-SOD. The total SOD activity in the beetle was negatively correlated to the O2•- content with a correlation coefficient of -0.437. An E. coli system was employed to study the function of each MpCu/Zn-SOD gene. The fusion proteins Trx-His-MpCu/Zn-SODs were over expressed in E. coli BL21 using pET32a vector, and identified by SDS-PAGE and Western blotting. The transformed bacteria BL21(pET32a-MpecCu/Zn-SOD) and BL21(pET32a-MpicCu/Zn-SOD) showed increased cold tolerance to -4 °C as well as increased SOD activity compared to the control BL21(pET32a). The relative conductivity and malondialdehyde content in the two MpCu/Zn-SODs transformed bacteria under -4 °C were significantly lower than the control BL21(pET32a). Furthermore, BL21(pET32a-MpecCu/Zn-SOD) had significantly higher SOD activity and cold tolerance than BL21(pET32a-MpicCu/Zn-SOD) under -4 °C treatment, and had lower conductivity than BL21(pET32a-MpicCu/Zn-SOD). In conclusion, low temperature led to the accumulation of O2•- in M. punctipennis, which stimulated the expression of extracellular MpCu/Zn-SOD gene and the increase of total SOD activity. E. coli overexpressing Trx-His-MpCu/Zn-SODs increased resistance to cold treatment-induced oxidative stress. Our findings will be helpful in further study of Cu/Zn-SOD genes in insect cold-tolerance.

Comparative analysis of the transcriptome of the overwintering desert beetle Microdera punctipennis.

The cold tolerance mechanisms of insect have been studied extensively on the model species Drosophila and a few other species at the transcriptional level. However studies on insects that inherit strong cold tolerance are limited. Cold hardy Tenebrionid beetle Microdera punctipennis is endemic to Gurbantonggut Desert, northwest of China. However, its genomic information is lacking. To investigate the overwintering mechanisms of M. punctipennis adult, RNA-seq was performed on the winter adults and the control adults that were kept in laboratory at 30 °C. A total of 175,247 unigenes were acquired with an average length of 645 bp. By using DESeq package, we identified 3367 unigenes that were up-regulated and 7988 down-regulated in the winter adults compared with the controls. To further our understanding of these differentially expressed genes (DEGs), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. Pathway analysis showed that the "ECM-receptor interaction", "PI3K-Akt signaling pathway", "Estrogen signaling pathway", "Tight junction", and "Regulation of actin cytoskeleton", etc. might play important roles in M. punctipennis overwintering. The DEGs results from the RNA-Seq were confirmed partially by qRT-PCR for 13 DEGs, which showed high consistence with a Pearson's correlation coefficient of 0.851. Overall, the sequence data will provide basic information for subsequent bioinformatical analysis and mining of the genes responsible for cold tolerance in M. punctipennis, as well as for understanding the molecular mechanisms of desert beetle overwintering.