Identification of target genes differentially expressed after pulse expression of Neisseria gonorrhoeae sRNAs

Purpose: The goal of this study was the identification of target genes of the Neisseria gonorrhoeae sRNAs NgncR_237, NgncR_162 and NgncR_163. Methods: RNA was isolated from N. gonorrhoeae using the miRNeasy Micro Kit (Qiagen). Enrichment of mRNA was done using the Universal Ribodepletion Kit followed by Next Ultra Directional Library Preparation Kit for Illumina (NEB). The cDNA was sequenced on Illumina HiSeq 3000 platform with 100 bp paired end reads. Adapters from Fastq sequences were removed using cutadapt version 1.2.1. Only reads exceeding a mean base quality 5 within all sliding windows of 5 bp were mapped to the genome of strain N. gonorrhoeae MS11 (reference genome ASM15685v2). Annotation of non-coding RNAs was according to Remmele et al.(2014). Read mapping was conducted using Bowtie2 v2.1.0. DeSEQ2 version 1.6.2 was used to identify differentially regulated transcripts. Results: N. gonorrhoeae genes were identified which were upregulated or downregulated upon induced expression of sRNAs NgncR_237, NgncR_162 and NgncR_163. Conclusions: sRNA NgncR_237, NgncR_162 and NgncR_163 are involved in the regulation of genes involved in type IV pilus biogenesis, energy metabolism and transport processes. RNAseq was performed on N. gonorrhoeae wild-type, sRNA knockout mutant Ng MS11 Δ237, a derivative of this strain expressing sRNA NgncR_237 under control of the Ptet promoter, sRNA double knockout mutant Ng MS11 ΔΔ162/163 and derivatives of this strain expressing sRNA NgncR_162 or NgncR_163 under control of the Ptet promoter. Strains were cultivated in the presence of anhydrous tetracycline for 30 min. Experiments were performed in triplicates.