Characterization of the Promoter, MxiE Box and 5′ UTR of Genes Controlled by the Activity of the Type III Secretion Apparatus in Shigella flexneri

Abstract

Activation of the type III secretion apparatus (T3SA) of Shigella flexneri , upon contact of the bacteria with host cells, and its deregulation, as in ipaB mutants, specifically increases transcription of a set of effector-encoding genes controlled by MxiE, an activator of the AraC family, and IpgC, the chaperone of the IpaB and IpaC translocators. Thirteen genes carried by the virulence plasmid ( ospB , ospC1 , ospD2 , ospD3 , ospE1 , ospE2 , ospF , ospG , virA , ipaH1.4 , ipaH4.5 , ipaH7.8 and ipaH9.8 ) and five genes carried by the chromosome ( ipaHa-e ) are regulated by the T3SA activity. A conserved 17-bp MxiE box is present 5′ of most of these genes. To characterize the promoter activity of these MxiE box-containing regions, similar ∼67-bp DNA fragments encompassing the MxiE box of 14 MxiE-regulated genes were cloned 5′ of lacZ in a promoter probe plasmid; β-galactosidase activity detected in wild-type and ipaB strains harboring these plasmids indicated that most MxiE box-carrying regions contain a promoter regulated by the T3SA activity and that the relative strengths of these promoters cover an eight-fold range. The various MxiE boxes exhibiting up to three differences as compared to the MxiE box consensus sequence were introduced into the ipaH9.8 promoter without affecting its activity, suggesting that they are equally efficient in promoter activation. In contrast, all nucleotides conserved among MxiE boxes were found to be involved in MxiE-dependent promoter activity. In addition, we present evidence that the 5′ UTRs of four MxiE-regulated genes enhance expression of the downstream gene, presumably by preventing degradation of the mRNA, and the 5′ UTRs of two other genes carry an ancillary promoter.

A Shigella flexneri Virulence Plasmid Encoded Factor Controls Production of Outer Membrane Vesicles

“…A smaller mutation that replaces approximately the middle third of ipaH9.8 with the scar sequence left behind after removal of tetRA is not polar ( Figure 1C Experiment 2). These results reflect mechanisms of S. flexneri gene regulation that are yet to be elucidated ( Bongrand et al 2012 ). The nonpolar ipaH9.8 mutant is included in the pWR100 collection, with and without tetRA , under the name “ ipaH9.8ΔNterm .” IpaH9.8 is an E3 ubiquitin ligase, and the catalytic ability of this Type III Secretion System effector depends on a cysteine residue found at amino acid position 337 ( Rohde et al 2007 ).…”

A Fluorescent Reporter Reveals On/Off Regulation of the Shigella Type III Secretion Apparatus during Entry and Cell-to-Cell Spread

“…Second, the antiactivator OspD1, which inhibits MxiE activity in resting conditions, is secreted with the prestored effectors (Parsot Cell Host & Microbe T3SA Activity in Shigella Flexneri et al, 2005). A MxiE:IpgC complex is then able to activate transcription of promoters carrying a MxiE box (Bongrand et al, 2012;Mavris et al, 2002b;Pilonieta and Munson, 2008). This dual control on MxiE activity ensures that the second set of effectors is produced only when the T3SA is active.…”

The synthesis of OspD3 (ShET2) inShigella flexneriis independent of OspC1

“…While the ospC1 promoter is still present in this construct, the ¡10 promoter sequence has been mapped to 237 bases upstream of the ospC1 translational start codon. 33 The ospC1 promoter region is therefore 1.98 kB upstream of the ospD3 translational start codon. Using this sequence information, we deleted the ospC1 promoter in both the wild-type and DospC1 mutant to verify that ospD3 transcription was still present (Fig.…”