Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes of Wild Type and invF Deficient Mutant of Pseudomonas fluorescens 2P24

InvF, an AraC-type transcriptional activator, regulated the expression of genes that encoded the secreted effector molecules SipABCD, SigD, SptP, and SopE in Salmonella typhimurium. However, it seems that almost all effector homologs were lost in P. fluorescens 2P24. To identify the InvF regulon in strain 2P24, the invF deficient mutant 2P24?invF was constructed, and we implemented transcriptome sequencing (RNA-seq). Both of strains 2P24 and 2P24?invF were incubated in mannitol-glutamate (MG) minimal medium at 28? for 6 h and 12 h. The cell fractions were separated by centrifugation at 10, 000 rpm for 5 min at 4°C. Total RNA was isolated using a E.Z.N.A.® Bacterial RNA Kit (Omega Bio-tek, USA) as described by the manufacturer for RNA-seq. mRNA profiles of strains 2P24 and 2P24?invF were generated by deep sequencing, in triplicate, using Illumina Hiseq. A total of 127 significantly differentially expressed genes (DEGs) were obtained by comparing the transcriptional level of strain 2P24?invF with 2P24 at 6 h and 12 h [Fold change = 1.5; p value = 0.05]. Fifty-two down-regulated genes were regarded as InvF regulon candidates. Among the InvF regulon candidates, 39 genes were screened based on their gene functional annotation for qRT-PCR verification. Finally, eight genes were confirmed to be regulated positively by InvF. They encoded dihydrolipoyl dehydrogenase, toxin-activating lysine-acyltransferase, phage infection protein, AsnC family protein, two hypothetical proteins, NAD(P)H dehydrogenase, and protein IolH, respectively. Overall design: mRNA profiles of strains 2P24 and 2P24?invF which were incubated in mannitol-glutamate (MG) minimal medium at 28? for 6 h and 12 h.