Regulation of the overlapping pic/set locus in Shigella flexneri and enteroaggregative Escherichia coli

Abstract

Most strains of Shigella flexneri 2a and enteroaggregative Escherichia coli carry a highly conserved chromosomal locus which encodes a 109-kDa secreted mucinase (called Pic) and, on the opposite strand in overlapping fashion, an oligomeric enterotoxin called ShET1, encoded by the setA and setB genes. Here, we characterize the genetic regulation of these overlapping genes. Our data suggest that pic and the setBA loci are transcribed as complementary 4-kb mRNA species. The major pic promoter is maximally activated at 37 degrees C in exponential growth phase. Our data suggest that the setB gene is transcribed from a promoter which lies more than 1.5 kb upstream of the setB structural gene; setA may be transcribed via readthrough of the setB transcript and possibly by its own promoter. The long leader of the setB gene provides a strong silencing effect on setB transcription. The signals which provide relief from setB silencing are not clear, but significant induction is observed in a continuous anaerobic culture of human fecal bacteria, suggesting that some complex characteristics of the human intestine act to lift repression of setB expression. Our studies provide the first insights into the mechanisms affecting expression of this unusual virulence locus.

FIG. 1.

Diagram of the apparatus used for continuous culture of human fecal bacteria. See Materials and Methods for a detailed description.

FIG. 2.

Organization of the pic/set locus. (A) Map of the chromosomal DNA fragment encoding pic and setBA and results of RT-PCR experiments to determine the initiation and termination sites of set transcription. Locations of nested set primers are indicated, along with presence (solid line) or absence (dotted line) of product generated by that primer pair. (B) Agarose gel electrophoresis demonstrating products of RT-PCR from the pic/set locus, using the primers illustrated in panel A. RNA was extracted from EAEC strain 042 and subjected to RT synthesis and cDNA amplification by standard PCR (cDNA lanes). A direct PCR step was carried out in parallel on 042 DNA to demonstrate the specificity of the PCR (DNA lanes). The primer pairs used are shown above the lanes. Molecular size markers are shown in the first lane of each gel; the sizes of some markers are indicated.

FIG. 3.

β-Galactosidase activity derived from pic and set promoter fusions either in vector pRS551 transformed into EAEC 042 or as single-copy chromosomal integrations in E. coli K-12 strain TE2680. Values are the results of triplicate determination + standard deviation. The plasmids are described in Table 2. The numbers above the inserts refer to nucleotide positions with respect to Fig. 2. pic fusions are expressed from left to right and set fusions from right to left with respect to the lacZ gene. ND, not determined.

FIG. 4.

DNA strand-specific regulation of the pic and set genes by the set DRE silencer region. Results represent β-galactosidase measurements of at least triplicate determinations from L-broth cultures grown to late logarithmic phase; error bars represent 1 standard deviation. Test strains were EAEC strain 042 transformed with pic or set promoter DNA cloned upstream of the lacZ reporter in plasmid pRS551. Constructs used in these experiments were pS37/19 (setB promoter with the silencer in the native orientation); pS37/19-inverted (setB promoter with the silencer in the inverted orientation), pP37/19 (P_pic2,3 promoter region with the silencer in the native orientation), and pP37/19-inverted (P_pic2,3 promoters with the silencer in the inverted orientation).

FIG. 5.

(A) Effect of growth phase on pic and set expression as determined by real-time quantitative RT-PCR. RNA was extracted from E. coli 042 grown in L-broth for various times (0.5, 1.5, and 6.5 h) after 1:20 dilution of an overnight culture with fresh medium. cDNA was synthesized and subjected to quantitative RT-PCR as described in Materials and Methods. (B) β-Galactosidase activity of E. coli 042 harboring plasmid pP15/2 (Ppic1,2,3), pP16/2 (Ppic2,3), or pP3/2 (Ppic3) grown in L-broth and assayed at various times after dilution of overnight cultures with fresh medium.

FIG. 6.

Transcriptional regulation of pic and set in EAEC strain 042, E. coli K-12, and S. flexneri 2457T. Plasmids containing all three pic promoter regions (P_pic1,2,3), two pic promoter regions (P_pic2,3), the most downstream pic promoter region (P_pic3), the setB promoter (P_setB), the setB promoter with the DRE silencer region, or the setA promoter (P_setA) constructed in pRS551 were transformed into EAEC 042, S. flexneri 2457T, or E. coli K-12 and tested for β-galactosidase activity. Bacterial strains were grown to exponential phase in L-broth medium at 37°C to the late logarithmic phase. Bars represent results of at least three determinations; error bars represent 1 standard deviation.