Enterohemorrhagic Escherichia coli (EHEC) is notable for the Shiga-like toxin and several secretion systems and virulence factors that facilitate intestinal colonization and pathogenesis. EHEC low infectious dose may be in part due to its Type VI secretion system (T6SS), which could have antibacterial activity and perhaps damage host cells, although its function and mechanism are unknown. The aim of this study was to understand T6SS activation in EHEC by replacing its unknown native promoter with two inducible promoters. We found that the structural components encoded in the tssB operon are expressed as part of a single polycistronic operon. Excitingly, in the other direction, the hcp3 region is formed of at least two separate operons, leading to incomplete T6SS activation and no secretion of T6SS substrates. Analysis of the region downstream of hcp3 revealed a transcriptional terminator, a promoter region, and multiple transcription factor binding sites; thereby, we designated it as h3R. This regulatory sequence represses the translation of vgrG3 but also contains a regulatory element that stabilizes the mRNA of hcp3, the gene located immediately upstream of vgrG3. Meanwhile, deletion of hns elevated T6SS mRNAs but was not sufficient for protein expression until further activated with DMEM or MM9, although not all proteins were expressed and protein secretion by other systems was modified. These data highlight the stringent regulatory control of the T6SS in EHEC and identify novel promoter regions that warrant further investigation to elucidate the role of this secretion system in bacterial competition and pathogenesis.IMPORTANCEEHEC harbors a T6SS, which in other bacteria play a role in intestinal colonization and pathogenesis. T6SS is repressed in vitro in EHEC, making its study difficult, but it has been suggested to be required for full pathogenesis of EHEC. Here, we aimed to activate the T6SS in vitro. We found that activation of the bidirectional promoter is not enough for full T6SS expression, as a regulatory sequence downstream, named h3R, tightly regulates its expression. Deletion of the master regulator hns was not enough for full activation, and additional activation was required to produce some, but not all, proteins, causing a loss of bacterial fitness. These findings bring us closer to a full understanding of the mechanisms that control its expression. We predict novel regulation sites that will be necessary to fully understand the activation mechanisms and, thus, the function of T6SS in EHEC and perhaps other bacteria.
Vazquez-Lopez et al. (Tue,) studied this question.