The Antiactivator of Type III Secretion, OspD1, Is Transcriptionally Regulated by VirB and H-NS from Remote Sequences in Shigella flexneri.

species, the causal agents of bacillary dysentery, use a type III secretion system (T3SS) to inject two waves of virulence proteins, known as effectors, into the colonic epithelium to subvert host cell machinery. Prior to host cell contact and secretion of the first wave of T3SS effectors, OspD1, an effector and antiactivator protein, prevents premature production of the second wave of effectors. Despite this important role, regulation of the gene is not well understood. While belongs to the large regulon of VirB, a transcriptional antisilencing protein that counters silencing mediated by the histone-like nucleoid structuring protein H-NS, it remains unclear if VirB directly or indirectly regulates Additionally, it is not known if is regulated by H-NS. Here, we identify the primary transcription start site (+1) and show that the promoter is remotely regulated by both VirB and H-NS. Our findings demonstrate that VirB regulation of requires at least one of the two newly identified VirB regulatory sites, centered at -978 and -1270 relative to the +1. Intriguingly, one of these sites lies on a 193-bp sequence found in three conserved locations on the large virulence plasmids of The region required for H-NS-dependent silencing of lies between -1120 and -820 relative to the +1. Thus, our study provides further evidence that -acting regulatory sequences for transcriptional antisilencers and silencers, such as VirB and H-NS, can lie far upstream of the canonical bacterial promoter region (i.e., -250 to +1). Transcriptional silencing and antisilencing mechanisms regulate virulence gene expression in many important bacterial pathogens. In species, plasmid-borne virulence genes, such as those encoding the type III secretion system (T3SS), are silenced by the histone-like nucleoid structuring protein H-NS and antisilenced by VirB. Previous work at the plasmid-borne locus revealed that VirB binds to a remotely located -acting regulatory site to relieve transcriptional silencing mediated by H-NS. Here, we characterize a second example of remote VirB antisilencing at , which encodes a T3SS antiactivator and effector. Our study highlights that remote transcriptional silencing and antisilencing occur more frequently in than previously thought, and it raises the possibility that long-range transcriptional regulation in bacteria is commonplace.