Two transcription factors and one antisense RNA underlie the thermoregulated insect pathogenicity of Yersinia entomophaga MH96

Entomopathogenic bacteria express virulence genes in a temperature-dependent manner, but the underlying molecular mechanisms remain poorly understood. Here, we describe the roles of an unusual LytTR-containing transcription factor Yen6 in determining the virulence of Yersinia entomophaga (MH96). An initial transcriptome analysis in vivo revealed that yen6, located 1,372 bp upstream from genes encoding the Yen Toxin complex (Yen-Tc), exhibited significantly higher transcriptional activity at 37 °C compared to 25 °C (adjusted p-value < 0.001) during intrahemocoelic infection in the insect model Galleria‍ mellonella. Deletion of yen6 also significantly reduced MH96's virulence in G.‍‍ mellonella by up to three-fold. Next, comparing the transcriptome of wild-type MH96 and its derived Δyen6 mutant revealed genes for ribose utilization were downregulated whereas genes for fructose utilization and the RNA-binding protein YhbY were upregulated in the yen6-deficient mutant. Subsequent electromobility shift assays showed that purified Yen6_His6 protein was capable of specifically binding to the promoter regions of the ribose and fructose utilization-related gene clusters, as well as yhbY. A 645-nucleotide-long 3' untranslated region, designated yen7_AS, was identified extending from the yen6 coding region. Notably, yen7_AS completely overlaps the yen7 gene on the opposing strand and may therefore act as a cis anti-sense RNA regulating yen7 expression, which encodes a putative transcriptional regulator of the Yen-Tc. This study highlights the critical role of the yen6-yen7 locus in MH96 virulence during G.‍ mellonella infection, including the complex mechanisms controlling insecticidal exoprotein production.