Exploration of the effects of a degS mutant on the growth of Vibrio cholerae and the global regulatory function of degS by RNA sequencing
Exploration of the effects of a degS mutant on the growth of Vibrio cholerae and the global regulatory function of degS by RNA sequencing
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通过RNA测序探讨degS突变体对霍乱弧菌生长的影响以及degS的整体调节功能
DOI：
10.7717/peerj.7959
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发表时间：
2019-10-23
期刊：
影响因子：
2.7
通讯作者：
Min, Xun
中科院分区：
文献类型：
--
作者：
Huang, Jian;Chen, Yuxi;Min, Xun
背景资料：DegS是一种周质丝氨酸蛋白酶，被认为是sigma（E）应激反应途径的启动子，该蛋白在大肠杆菌应激反应的调节中发挥着重要作用。杆菌然而，对DegS在霍乱弧菌中的生物学功能和全球调控网络的了解仍然有限。本研究的目的是研究degS在霍乱弧菌HN 375中的分子功能和调控网络。通过平板生长实验观察细菌菌落形态，通过生长曲线实验观察细菌生长能力。采用高通量RNA测序（RNA-Seq）技术分析野生型和degS突变株之间的差异转录组学谱。采用基因本体论、通路分析和基因行为网络分析等方法对差异表达基因的主要功能进行了研究。进行定量实时PCR（qRT-PCR）以验证RNA-Seq分析的可靠性和准确性。结果：degS缺失后，degS突变株在各种培养基上的菌落数均小于野生型菌株，生长速度也比野生型菌株慢。共鉴定出423个差异表达基因，包括与野生型菌株相比在degS突变体中上调的187个基因和相对下调的236个基因。GO分类和途径分析表明，许多差异表达的基因与各种细胞代谢途径和细胞周期有关。此外，Gene-Act网络分析表明，许多差异表达的基因参与细胞代谢途径和细菌的趋化性。cAMP-CRP-RpoS信号通路和LuxPQ信号转导系统也受到degS突变体的影响。随机选择的9个差异表达基因的表达模式在qRT-PCR和RNA-seq结果之间一致。互补实验表明，pBAD 24-degS或pBAD 24-rpoS质粒可部分恢复小菌落degS突变表型。这些差异表达的基因可能与细菌的生长有关，涉及多种细胞代谢过程和细胞周期。几个新的degS相关的监管网络被确定。此外，我们的研究结果表明，cAMP-CRP-RpoS信号通路可能参与了小菌落degS突变表型。总之，我们相信这些转录组学数据将为研究霍乱弧菌degS的功能提供有用的遗传资源。
Background: DegS is a periplasmic serine protease that is considered to be the initiator of the sigma(E) stress response pathway, and this protein plays an important role in the regulation of the stress response in E. coli. However, knowledge of the biological function and global regulatory network of DegS in Vibrio cholerae remains limited. In this study, we aimed to characterize the molecular functions and further investigate the regulatory network of degS in V. cholerae.Methods: A deletion mutant of degS was constructed in the V. cholerae HN375 strain. Bacterial colony morphology was observed by a plate-based growth experiment, and bacterial growth ability was observed by a growth curve experiment. High-throughput RNA sequencing (RNA-Seq) technology was used to analyze the differential transcriptomic profiles between the wild-type and degS mutant strains. Gene ontology (GO), pathway analysis and Gene-Act-network analysis were performed to explore the main functions of the differentially expressed genes. Quantitative real-time PCR (qRT-PCR) was performed to validate the reliability and accuracy of the RNA-Seq analysis. The complementation experiments were used to test the roles of degS and ropS in the small colony degS mutant phenotype.Results: When degS was deleted, the degS mutant exhibited smaller colonies on various media and slower growth than the wild-type strain. A total of 423 differentially expressed genes were identified, including 187 genes that were upregulated in the degS mutant compared to the wild-type strain and 236 genes that were relatively downregulated. GO categories and pathway analysis showed that many differentially expressed genes were associated with various cellular metabolic pathways and the cell cycle. Furthermore, Gene-Act network analysis showed that many differentially expressed genes were involved in cellular metabolic pathways and bacterial chemotaxis. The cAMP-CRP-RpoS signaling pathway and the LuxPQ signal transduction system were also affected by the degS mutant. The expression patterns of nine randomly selected differentially expressed genes were consistent between the qRT-PCR and RNA-seq results. The complementation experiments showed that the small colony degS mutant phenotype could be partially restored by complementation with the pBAD24-degS or pBAD24-rpoS plasmid.Discussion: These results suggest that the degS gene is important for normal growth of V. cholerae. Some of the differentially expressed genes were involved in various cellular metabolic processes and the cell cycle, which may be associated with bacterial growth. Several new degS-related regulatory networks were identified. In addition, our results suggested that the cAMP-CRP-RpoS signaling pathway may be involved in the small colony degS mutant phenotype. Overall, we believe that these transcriptomic data will serve as useful genetic resources for research on the functions of degS in V. cholerae.