Complex regulation of csgD promoter activity by global regulatory proteins

Abstract

SummaryThe starvation-induced csgD gene of Salmonella typhimurium encodes for the positive transcriptional regulator of extracellular matrix components curli fimbriae and cellulose. To analyse regulatory elements of csgD promoter ( PcsgD ) response genetic studies combined with in vitro experiments were performed. Six binding sites (D1 to D6) for OmpR, a transcriptional regulator, were identified by gel shifts and DNase I footprints. While ompR is required for PcsgD expression, binding of OmpR-P to D2 centred immediately upstream of D1 at position ----70.5 is proposed to repress PcsgD activity. The elevated expression of regulated and semiconstitutive PcsgD in response to microaerophilic conditions required integration host factor (IHF). Subsequently, two IHF-binding sites were identified up-and downstream of PcsgD . IHF competes with OmpR-P for binding at its upstream site IHF1, which overlaps with D3-D6 and thereby modulates the response to microaerophilic conditions. A complex regulatory network involving IHF, H-NS and OmpR is proposed whereby the nucleo-complex composition in the csgD -csgBA intergenic region is altered in response to oxygen tension.

Comparative Genetics of the rdar Morphotype inSalmonella

“…To analyze the genetic conservation of Tafi production throughout the salmonellae, the agfDEFG-agfBAC intergenic region was amplified from each SARC16 isolate and used to generate agfD and agfB promoter luciferase fusions (7). This DNA region included the 521-bp intertranscript region and 5Ј untranslated regions of agfB and agfD (23). We were unable to amplify agfD-agfB fragments from S. enterica subsp.…”

“…For PagfD, the S. enterica subsp. arizonae isolates both possessed three unique changes in the activating OmpR binding site required for agfD transcription (23,52) (Fig. 3).…”

“…3). Two of the changes were in the most highly conserved nucleotides in the OmpR consensus binding sequence (ACNTTTNGNTACANNTAT) (23,33).…”

“…Each isolate acquired mutations that elevated agfD expression, either directly through cis mutations in the PagfD region (Sarc4 and Sarc16) or through trans regulatory changes upstream of agfD (Sarc11) ( Table 3). The nucleotide change in Sarc4 was at the same position as the PagfD change in SE 3b (52) and may represent a mutational hotspot within the OmpR binding region (ACNTTTNGNTACANNTAT) (23). For Sarc16, the change was in the Ϫ35 region and shifted the PagfD sequence closer to the 70 consensus (39).…”

“…trans-acting regulatory proteins required for agfD transcription include OmpR and MlrA. The arrangement of OmpR binding sites in the agfD promoter region is similar to that in the well-characterized ompF promoter (33), with a high-affinity binding site for activation under conditions of low osmolarity and low-affinity binding sites that shut off transcription at higher osmolarity (23). The mechanism for MlrA activation of agfD transcription is unknown, and no binding sites have been identified (13).…”