Purpose:To investigate the effect of small RNA RyhB-1and RyhB-2 deletion of Salmonella enteritidis on genes expression of genome under simulated intestinal conditions
Methods:S. Enteritidis wild type (WT) strain 50336 and mutants △ryhB-1, △ryhB-2, and △ryhB-1/ryhB-2 were grown overnight in 50 mL of the LB medium with 160 rpm shaking at 37°C under aerobic condition, washed thrice, and re-suspended with iron-limited and nutrient-limited medium. The above prepared bacterial cells were cultured for 2 h at 37°C in the anaerobic workstation. Each sample was analysed in triplicate. Total RNA of the above stains was extracted using TRIzol reagentwith DNase digestion according to the manufacturer’s instruction. Ribosome RNA was removed using a Ribo-Zero rRNA removal kit (Illumina) that leaves the mRNA. Sequencing libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB), sequenced on an Illumina Hiseq 2000 platform at Beijing Novogene Bioinformatics Technology。
Results: Analysis of the RNA-Seq data identified 496, 250, and 553 DEGs in △ryhB-1, △ryhB-2, and △ryhB-1△ryhB-2, respectively, compared to the WT strain.A total of 213 genes changed in △ryhB-1, △ryhB-2, and △ryhB-1△ryhB-2 mutants. The transcriptional levels of 176 genes changed in △ryhB-1 and △ryhB-1△ryhB-2 but not in △ryhB-2. Furthermore, 14 genes were only changed in RyhB-2, 96 DEGs were only identified in △ryhB-1, and 12 DEGs were only identified in △ryhB-2 but not in △ryhB-1△ryhB-2. Moreover, 150 DEGs were only identified in △ryhB-1△ryhB-2 but neither in △ryhB-1 nor in △ryhB-2.
Conclusions: A total of 213 genes could be regulated by △ryhB-1, △ryhB-2, and △ryhB-1△ryhB-2 mutants, 176 genes are regulated only by RyhB-1, 14 genes were regulated only by RyhB-2.
Overall design: Including 4 samples, Wild Type, ryhB-1, ryhB-2, ryhB-1/ryhB-2 Mutants, and each sample was analysed in triplicate. The RNA-Seq clean reads were aligned to the Salmonella Enteritidis str. P125109 genome from NCBI (https://www.ncbi.nlm.nih.gov/nuccore/NC_011294.1) using Bowtie2-2.2.3 (51). Rockhopper [13,14] was used for RNA-Seq data analysis (https://cs.wellesley.edu/~btjaden/Rockhopper/index.html), including differential gene expression detection, novel and reference-based transcript identification, and operon prediction. Gene expression was quantified as reads per kilobase of coding sequence per million reads (RPKM) algorithm. Genes with an adjusted q values < 0.01 and [log2(fold change)] >1 were assigned as differentially expressed genes (DEGs).
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