Ler promotes the expression of many H-NS-repressed SYN-117 virulence genes including those of LEE1-5, grlRA and non-LEE-encoded virulence genes such as lpf and the virulence plasmid pO157-encoded mucinase stcE[26, 28, 31, 36–39]. Thus, Ler antagonizes H-NS in the regulation of many virulence genes, which belong to both the H-NS and Ler (H-NS/Ler) regulons. The E. coli stringent starvation protein A (SspA) is a RNA polymerase-associated protein mTOR inhibitor cancer [40] that is required for transcriptional activation of bacteriophage P1 late genes and

is important for survival of E. coli K-12 during nutrient depletion and prolonged stationary phase [41–43]. Importantly, SspA down-regulates the cellular H-NS level during stationary phase, and thereby derepress the H-NS regulon including genes

for stationary phase induced acid tolerance in E. coli K-12 [44]. A conserved surface-exposed pocket of SspA is important for its activity as a triple alanine substitution P84A/H85A/P86A in surface pocket residues abolishes SspA activity [45]. SspA is highly conserved among Gram-negative pathogens [44], which suggests a role of SspA in bacterial pathogenesis. Indeed, SspA orthologs affect the virulence of Yersinia enterocolitica, Neisseria gonorrhoeae, Vibrio cholerae, Francisella tularensis and Francisella novicida[46–51]. Since E. coli K-12 SspA is conserved in EHEC where H-NS negatively Tanespimycin modulates virulence gene expression, we asked the question of whether SspA-mediated regulation of H-NS affects EHEC virulence gene expression. Here we study the effect of SspA on the expression of LEE- and non-LEE-encoded virulence genes and its effect on H-NS

accumulation in EHEC. Our results show that in an sspA mutant elevated levels of H-NS repress the expression of virulence genes encoding the T3SS system rendering the cells incapable of forming A/E lesions. 3-mercaptopyruvate sulfurtransferase Thus, our data indicate that SspA positively regulates stationary phase-induced expression of H-NS-controlled virulence genes in EHEC by restricting the H-NS level. Results and discussion SspA positively affects transcription of EHEC virulence genes To evaluate the effect of sspA on virulence gene expression in EHEC during the stationary phase we constructed an in-frame deletion of sspA in the E. coli O157:H7 strain EDL933 ATCC 700927 [52] and measured transcription of LEE- (LEE1-5, grlRA and map) and non-LEE-encoded (stcE encoded by pO157) genes (Figure  1). Wild type and sspA mutant strains were grown in LB medium to stationary phase with similar growth rates (data not shown). Total RNA was isolated and transcript abundance was measured by primer extension analyses using labeled DNA oligos specific to each transcript of interest and ompA, which served as internal control for total RNA levels.