Exploration of the effects of a degS mutant on the growth of Vibrio cholerae and the global regulatory function of degS by RNA sequencing
Authors: Huang Jian , Chen Yuxi , Chen Jie , Liu Changjin , Zhang Tao , Luo Shilu , Huang Meirong , Min Xun 
Published: October 2019

Background
DegS is a periplasmic serine protease that is considered to be the initiator of the σE 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.