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

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

Potential secondary structure involving the sequence encoded by the KpnI-HindIII fragment of pQF50 and the <i>lacZ</i> ribosome binding site.

<p>The potential secondary structure of the 5′ UTR mRNA arising from the minimal <i>ipaH9.8</i> promoter cloned at the KpnI site of pQF50 is presented, with nucleotides specific of <i>ipaH9.8</i> shown in grey, the KpnI site in red, the HindIII site in green and the ribosome-binding site (RBS) and translation start codon of <i>lacZ</i> in blue characters.</p

Minimal promoter regions cloned into pQF50.

<p>Sequences are aligned with respect to the MxiE and −10 boxes; nucleotides matching the consensus sequence of the MxiE box and the proposed −10 box are shown in uppercase characters. Nucleotides differing from the MxiE box and −10 box consensus sequences are shown in red. Nucleotides differing between the <i>ospE1</i> and <i>ospE2</i> promoter regions are highlighted in blue and nucleotides differing between the <i>ipaH9.8</i> and <i>ipaHc</i> promoter regions are highlighted in yellow. Each of these sequences was inserted between the NcoI (in 5′) and KpnI (in 3′) sites of pQF50.</p

Effects of mutations in conserved positions of the MxiE box on the <i>ipaH9.8</i> promoter activity.

<p>β-galactosidase activity was assayed in derivatives of the wild-type (open bars) and <i>ipaB</i> (filled bars) strains harboring a plasmid carrying the <i>ipaH9.8</i> promoter (from −57 to +85) with the mutation in the MxiE box indicated below the bars. Activities are expressed in Miller units.</p

MxiE box-containing regions located 5′ of genes controlled by the T3SA activity.

<p>Sequences located 5′ to the translation start site (ATG) of MxiE-regulated genes are aligned with respect to the MxiE box and proposed −10 box and transcription start site (+1). Nucleotides identical to those present in the consensus sequence of the MxiE and −10 boxes (top row) are shown in bold uppercase characters. The number of nucleotides present in the 5′ UTR (from the nucleotide in position +1 to the nucleotide 5′ of the translation start site) is indicated.</p

Expression of <i>lacZ</i> from plasmids carrying 5′ UTRs inserted 3′ of the <i>lac</i> promoter.

<p>β-galactosidase activity was assayed in derivatives of the wild-type strain harboring plasmids carrying the 5′ UTR of genes indicated below the bars inserted between the <i>lac</i> promoter and <i>lacZ</i>. The cloned 5′ UTRs extend from position +2 (with respect to the transcription start site) to position −13 (with respect to the translation start site) of genes indicated below the bars. Activities are expressed in Miller units.</p

Expression of <i>lacZ</i> from plasmids carrying the <i>ipaH9.8</i> promoter with 5′ UTRs of different lengths.

<p>β-galactosidase activity was assayed in derivatives of the wild-type (open bars) and <i>ipaB</i> (filled bars) strains harboring plasmids carrying the <i>ipaH9.8</i> promoter with 5′ UTRs of different lengths. Numbers below the bars indicate the position of the 3′ end of the cloned 5′ UTR. Activities are expressed in Miller units.</p

Potential secondary structures of the mRNA in the 5′ UTR of <i>ipaH9.8</i> and <i>ipaHc</i>.

<p>Potential secondary structures of the mRNA in the 5′ UTR of <i>ipaH9.8</i> and <i>ipaHc</i>.</p

Effects of variations in the MxiE box on the <i>ipaH9.8</i> promoter activity.

<p>A: the MxiE box consensus sequence is shown on the upper row; only nucleotides differing from the consensus sequence in various MxiE boxes are indicated below, together with the name of the gene controlled by this MxiE box. Nucleotides corresponding to position 15 (indicated as n in the consensus sequence) are indicated in lower cases. MxiE boxes of <i>ipaHc</i>, <i>ipaHe</i>, <i>ipaH7.8</i>, <i>ospE1</i> and <i>ospE2</i> (being identical to the one of <i>ipaH9.8</i>) and the MxiE box of <i>ospC1</i> (differing from that of <i>ipaH9.8</i> only at position 15) are not shown. B: β-galactosidase activities were assayed in derivatives of the wild-type (open bars) and <i>ipaB</i> (filled bars) strains harboring a plasmid carrying the <i>ipaH9.8</i> promoter (from −57 to +85) with the MxiE box of genes indicated below the bars. Activities are expressed in Miller units.</p

Annotations of chromosomal <i>ipaH</i> genes in <i>Shigella</i> genomes^a.

a<p>Names used to annotate the chromosomal <i>ipaH</i> genes in different genomes are indicated; in this study, we used the letter-based nomenclature indicated in the left column. Genes inactivated by a frameshift mutation or an insertion sequence are indicated (fs) and (IS), respectively, and genes absent from a genome are indicated by a hyphen.</p