Exploration of the effects of a degS mutant on the growth of Vibrio cholerae and the global regulatory function of degS by RNA sequencing

Strain cultivation and DNA manipulation

The V. cholerae HN375 strain is a non-O1/non-O139 strain collected by the China Center for Type Culture Collection with accession number CCTCC AB209168 (Luo et al., 2011). This strain was used as the wild-type strain in this study. V. cholerae was cultured in Luria Bertani (LB) liquid medium with shaking or on LB agar plates at 37 °C. The degS in-frame deletion mutants were generated by the suicide vector pWM91 as described previously (Metcalf et al., 1996). To complement the expression of degS and rpoS in degS mutant strains, the degS gene and rpoS gene fragments were amplified and then ligated into the expression vector pBAD24 (Guzman et al., 1995). The pBAD24-degS and pBAD24-rpoS plasmid were transformed into the V. cholerae degS mutant, respectively. Subsequently, these strains were cultured in LB medium supplemented with 0.1% arabinose to induce gene expression.

Bacterial growth experiments

The V. cholerae HN375 wild-type and degS deletion mutant strains were cultured on Columbia blood agar plates for 16 h at 37 °C. For the plate-based growth experiment, bacteria were picked and inoculated onto blood agar, thiosulfate citrate bile salts sucrose (TCBS) agar, MacConkey agar and LB agar plates using the sectional streak method, and the bacterial morphology was observed after 24 h. The complement strains cultured in LB medium supplemented with 0.1% arabinose, the plate-based growth experiment was performed as described above. Colony size was measured by image J software. The significant differences in the colony diameters were performed with independent-sample t-tests using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). For the growth curve experiment, a 0.5 McFarland’s standard of fresh bacterial suspension was prepared. The 100-μl bacterial suspensions were seeded into 100 ml of fresh LB liquid medium with shaking at 37 °C, and the OD₆₀₀ values were recorded once per hour. Three technical replicates were performed for each strain. The above experiments were repeated independently three times. The significant differences in the growth curve assays were determined with two-way ANOVA using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA).

RNA-Seq data analysis

For in vitro RNA preparation, the wild-type and degS deletion mutant strains were grown to mid-exponential phase (OD₆₀₀ of ~0.6) in LB liquid medium. Total RNA was extracted from the wild-type and degS deletion mutant using TRIzol reagent (Takara, Dalian, China). After quality control, the cDNA libraries were prepared, and sequencing was performed by NovelBio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Raw reads were generated and filtered. The clean reads were used for mapping to the V. cholerae N16961 reference genome (RefSeq accession numbers NC_002505 and NC_002506) using Misplacing software (Wang et al., 2010). Gene expression was determined and normalized using the reads per kilobase per million (Mortazavi et al., 2008). Differential expression between the degS deletion mutant and wild-type strains was identified by the DEseq package (Audic & Claverie, 1997). After statistical analysis, the differentially expressed genes were defined according to fold change >1.5 or fold change <0.667 and the false discovery rate (FDR) threshold (FDR < 0.05).

Gene ontology and pathway analyses

To analyze the main functions of the differentially expressed genes, the differentially expressed genes corresponding to GO terms in the GO database were mapped using the GO-Term Finder tool (Boyle et al., 2004). The differentially expressed genes were annotated in the following three main categories based on the database analysis: biological processes, molecular functions and cellular components. Significantly enriched GO terms were limited to categories with P-value < 0.05.

To identify significantly enriched pathways, pathway annotations of the differentially expressed genes were performed according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Aoki-Kinoshita & Kanehisa, 2007). The significantly enriched pathways were selected based on Fisher’s exact test and were defined as having P-value < 0.05.

Gene-Act network

The Gene-Act network was established according to the gene–gene relationships in the KEGG database, including activation, binding, expression, inhibition and compound (Binder & Schumacher, 2008; Wang et al., 2013), which helped identify the main signaling pathways and key regulatory genes.

Validation of RNA-Seq data by qRT-PCR

To validate the RNA-Seq data, Quantitative real-time PCR (qRT-PCR) was performed to determine the mRNA expression level. Briefly, total RNA from the wild-type and degS deletion mutant strains was used for cDNA synthesis using the PrimeScript RT Reagent Kit (Takara, Dalian, China). The qRT-PCR assays were performed using the CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) and SYBR Premix Ex Taq Kit (Takara, Dalian, China). The 16s RNA was used as an endogenous reference gene, and the relative changes in gene expression were calculated based on the 2^−ΔΔCt method. The primer sequences used are shown in Table S1. Comparisons among groups were performed with independent-sample t-tests using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA).

The degS gene is important for normal growth of V. cholerae

To explore the biological functions of degS in V. cholerae, we constructed a deletion mutant of degS in the V. cholerae HN375 strain. The colonies of the degS mutant on blood agar, TCBS agar, MacConkey agar and LB agar plates were consistently smaller than those of the wild-type strain (Figs. 1A–1I). Next, we compared the bacterial growth curves of the degS mutant and wild-type strains. The degS mutant has a longer log phase than the wild-type strain (Fig. 1J). After entering the exponential growth phase, the wild-type and degS mutant have similar growth rate, while in the stationary phase, the density of degS mutant was lower than that of the wild-type strain. Together, these results suggest that degS is important for normal growth of V. cholerae.

Analysis of differential gene expression

To explore the molecular changes in the response to the degS deletion mutant in V. cholerae, we performed a comparative gene expression analysis between the degS deletion mutant and wild-type strains using RNA-Seq. After sequencing and data analysis, the differentially expressed genes were identified using the following criteria: fold change in expression >1.5 or <0.667 and FDR < 0.05. In total, 423 differentially expressed genes were identified (Fig. 2A), including 187 upregulated genes and 236 downregulated genes (Fig. 2B; Table S2). To our knowledge, some of the genes associated with energy metabolism and cell division were also found in this analysis, such as VC0485 (pyruvate kinase), VC2738 (phosphoenolpyruvate carboxykinase), VCA0843 (glyceraldehyde-3-phosphate dehydrogenase) and murB, which may affect bacterial growth. Together, our results show distinct gene profiles between the wild-type and degS mutant strains, suggesting that the degS deletion mutant can produce unique gene profiles that may be associated with V. cholerae growth.

GO analysis of differentially expressed genes

To understand the main functions of the differentially expressed genes, GO analysis was performed according to the GO project. Based on our RNA-Seq data, the primary GO categories for the downregulated GO terms were focused on transmembrane transport, oligopeptide transmembrane transport, lipid metabolic process, glucose metabolic process, negative regulation of phosphate metabolic process, regulation of cell division and others (Fig. 3A). Notably, the GO categories include the lipid metabolic process, glucose metabolic process, negative regulation of the phosphate metabolic process, leucine metabolic process and generation of precursor metabolites and energy, which are related to cellular metabolism. In addition, we found that the GO term categories for the regulation of cell division, regulation of cell septum assembly and regulation of the cell cycle were associated with bacterial growth. The main GO terms for the upregulated genes were the cellular response to iron ion, intracellular protein transmembrane transport, coenzyme A biosynthetic process and others (Fig. 3B).

Pathway analysis

To identify the significant pathways, KEGG enrichment pathway analysis was performed based on the KEGG database. The results showed that the 423 differentially expressed genes were mapped to 65 pathways. The top 20 enriched pathways are shown in Fig. 4. Overall, seven statistically significant enriched pathways were defined by the hypergeometric test (Table 1). The statistically significant enriched pathways were mainly involved in glycolysis/gluconeogenesis; carbon metabolism; glycine, serine and threonine metabolism; the citrate cycle (TCA cycle); energy metabolism; valine, leucine and isoleucine degradation; and pyruvate metabolism. These results suggest that there is a regulatory relationship between degS and cellular metabolic pathways.

Gene-Act network analysis

To further explore the relationships among these differentially expressed genes, a Gene-Act network was constructed based on the genes and genetic relationships, including activation, binding, expression, inhibition and compounds. According to the Gene-Act network shown in Fig. 5, important network modules, including VC2738 (phosphoenolpyruvate carboxykinase), VC2646 (phosphoenolpyruvate carboxylase), VC0485 (pyruvate kinase), VC0432 (malate dehydrogenase), VC1304 (fumarate hydratase, class I), VC2090 (the succinate dehydrogenase hydrophobic membrane anchor protein), VC0794 (hypothetical protein) and VCA0192 (D-lactate dehydrogenase), were involved in multiple cellular metabolic pathways, as mentioned in Table 1 above. We also found that VCA0278 (serine hydroxymethyltransferase), VCA0666 (L-serine dehydratase 1), VCA0875 (D-serine dehydratase), VC0027 (threonine dehydratase) and VC2638 (dihydrolipoamide dehydrogenase) were involved in glycine, serine and threonine metabolism. Interestingly, the interaction of two important genes, namely, VCA0737 (the luxP protein) and VCA0736 (the sensor histidine kinase LuxQ), in the quorum sensing system and the interaction of two important genes, namely, VC2614 (the cAMP-regulatory protein) and VC0534 (the RNA polymerase sigma-38 factor, rpoS), in the cAMP-CRP-RpoS signaling pathway, were also observed in the gene interaction network. In addition, four genes, namely, VCA0008 (amethyl-accepting chemotaxis protein), VC2201 (the chemotaxis protein CheR), VC1643 (amethyl-accepting chemotaxis protein) and VC2062 (achemotaxis-specific methylesterase), contribute to the bacterial chemotaxis pathway and were also affected by the degS mutant.

Validation of the RNA-Seq data

To validate the reliability and accuracy of the RNA-Seq analysis, nine differentially expressed genes were randomly selected for validation by qRT-PCR. As shown in Fig. 6, compared with the V. cholerae wild-type strain, the differentially expressed genes VCA0875, VC2036, VC2646, VC0485, VCA0008 and VC0736 were significantly upregulated, while VC2738, VC0534 and VCA0843 were significantly downregulated, in the degS mutant strain. These results were consistent with those of the RNA-Seq analysis, indicating the reliability and accuracy of the RNA-Seq data.

The small colony degS mutant phenotype is rescued by supplementing the expression of degS and rpoS gene

To further test the role of degS in the small colony phenotype, we constructed pBAD24-degS plasmid to complement degS gene in degS mutant strains. The small colony phenotype could be restored in degS mutants by complementation with the pBAD24-degS plasmid (Figs. 7A–7F). According to the above Gene-Act network, the cAMP-CRP-RpoS signaling pathway was significantly inhibited in the degS deletion mutant. We further confirmed that the expression of rpoS was inhibited in degS mutants and restored in degS mutants by complementation with the pBAD24-degS plasmid (Fig. 7G). RpoS has been reported to aid bacterial growth and survive in stationary phase (McCann, Kidwell & Matin, 1991; Shah et al., 2019). Therefore, to test whether rpoS is involved in the small colony degS mutant phenotype, we constructed pBAD24-rpoS plasmid to supplement the expression of ropS gene in degS mutant strains. The results showed that the small colony phenotype could be partially restored by supplement the expression of ropS gene.