Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Dec;187(23):7918-30.
doi: 10.1128/JB.187.23.7918-7930.2005.

A positive regulatory loop controls expression of the locus of enterocyte effacement-encoded regulators Ler and GrlA

Jeannette Barba  1 , Víctor H Bustamante, Mario A Flores-Valdez, Wanyin Deng, B Brett Finlay, José L Puente
Affiliations

A positive regulatory loop controls expression of the locus of enterocyte effacement-encoded regulators Ler and GrlA

Jeannette Barba et al. J Bacteriol. 2005 Dec.

Abstract

The formation of attaching and effacing (A/E) lesions on intestinal epithelial cells is an essential step in the pathogenesis of human enteropathogenic and enterohemorrhagic Escherichia coli and of the mouse pathogen Citrobacter rodentium. The genes required for the development of the A/E phenotype are located within a pathogenicity island known as the locus of enterocyte effacement (LEE). The LEE-encoded transcriptional regulators Ler, an H-NS-like protein, and GrlA, a member of a novel family of transcriptional activators, positively control the expression of the genes located in the LEE and their corresponding virulence. In this study, we used C. rodentium as a model to study the mechanisms controlling the expression of Ler and GrlA. By deletion analysis of the ler and grlRA regulatory regions and complementation experiments, negative and positive cis-acting regulatory motifs were identified that are essential for the regulation of both genes. This analysis confirmed that GrlA is required for the activation of ler, but it also showed that Ler is required for the expression of grlRA, revealing a novel regulatory loop controlling the optimal expression of virulence genes in A/E pathogens. Furthermore, our results indicate that Ler and GrlA induce the expression of each other by, at least in part, counteracting the repression mediated by H-NS. However, whereas GrlA is still required for the optimal expression of ler even in the absence of H-NS, Ler is not needed for the expression of grlRA in the absence of H-NS. This type of transcriptional positive regulatory loop represents a novel mechanism in pathogenic bacteria that is likely required to maintain an appropriate spatiotemporal transcriptional response during infection.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Expression of C. rodentium ler is regulated by global and specific regulators. (A) Schematic representation of the ler regulatory region. The bent arrow indicates the previously reported transcriptional start site (+1) (12). −35 and −10 consensus sequences are shown as black boxes. A large hatched box represents the insertion sequence element (IS679) localized at the 5′ end of the C. rodentium LEE (11). Open and hatched boxes indicate the approximate positions of negative and positive regulatory sequences (NRS and PRS), respectively, revealed by expression analysis of ler-cat transcriptional fusions. The PRS contains the putative IHF binding site. A gray box indicates a region required for GrlA and H-NS-mediated regulation of ler. Schematic representations of the ler-cat transcriptional fusions are shown below the diagram of the ler regulatory region. The ler-cat fusions were named pCRler and numbered according to the position of the 5′ end of the ler region contained in each fusion with respect to the transcriptional start site. (B) Expression of the ler-cat fusions was monitored in C. rodentium DBS100, E. coli MC4100, and E. coli MC4100 Δhns. The CAT specific activity was determined from samples collected from bacterial cultures grown for 6 h in DMEM at 37°C without agitation in a 5% CO2 atmosphere. The values are the means of at least three independent experiments performed in duplicate. Standard errors are shown with error bars.
FIG. 2.
FIG. 2.
GrlA is required for expression of C. rodentium ler. The expression of representative ler-cat fusions was monitored in C. rodentium DBS100, C. rodentium Δler, and C. rodentium ΔgrlA (A) or in E. coli MC4100 containing the pMPM-T3 vector or its derivative pTCRLer4 or pTCRGrlA1, expressing Ler or GrlA, respectively. As a control, the expression of a LEE2-cat fusion (pLEE2-CAT) was analyzed in the same strains (B). The expression of representative ler-cat fusions was monitored in E. coli MC4100 and its isogenic hns mutant containing plasmid pMPM-T3 or pTCRGrlA1 (C). The CAT specific activity was determined as described for Fig. 1. The values are the means of at least three independent experiments performed in duplicate. Standard errors are shown with error bars.
FIG. 3.
FIG. 3.
Ler is required for C. rodentium grlRA expression. (A) Schematic representation of the rorf3-grlRA region. Hatched boxes indicate negative regulatory sequences (NRS) revealed by expression analysis of the grlRA-cat transcriptional fusions. The bent arrow indicates the transcriptional start site (+1) for grlRA determined in this study. Schematic representations of the grlRA-cat transcriptional fusions are shown below the diagram of the rorf3-grlRA region. The positions for the 5′ and 3′ ends of the rorf3-grlRA region contained in each fusion, with respect to the transcriptional start site of grlRA, are shown to the left of the fusions. The grlRA-cat fusions were named pCRgrlRA and numbered consecutively as shown at the right of the diagram. (B) Expression of the grlRA-cat fusions was monitored in C. rodentium DBS100 and its isogenic ler and grlA mutants. The CAT specific activity was determined as described for Fig. 1. The values are the means of at least three independent experiments performed in duplicate. Standard errors are shown with error bars.
FIG. 4.
FIG. 4.
Primer extension analysis of the C. rodentium grlRA promoter region. (A) Total RNAs were obtained from culture samples of C. rodentium wild-type (WT), Δler, and ΔgrlA strains grown for 6 h in DMEM at 37°C without agitation in a 5% CO2 atmosphere. Primer extension assays were performed with purified total RNA and a primer specific for the grlR structural gene or a primer specific for ompA, which was used as a control. (B) Sequence of the intergenic region between rorf3 and grlRA. The transcriptional start site (+1) and the −10 and −35 promoter sequences for grlRA are shown with bold underlined letters.
FIG. 5.
FIG. 5.
Ler activates and H-NS represses the expression of grlRA-cat fusions in an E. coli K-12 strain. (A) The expression of representative grlRA-cat fusions was monitored in E. coli MC4100 containing plasmid pMPM-T3 (vector), pTCRLer4, or pTCRGrlA1. As a control, the expression of pLEE2-CAT was analyzed in the same strains. (B) H-NS mediates repression of grlRA expression. The expression of grlRA-cat fusions was monitored in E. coli MC4100 and its isogenic hns mutant containing plasmid pMPM-T3 (vector) or pTCRLer4 (ler). The CAT specific activity was determined as described for Fig. 1. The values are the means of at least three independent experiments performed in duplicate. Standard errors are shown with error bars.
FIG. 6.
FIG. 6.
Binding of Ler and H-NS to the rorf3-grlRA region. (A) Schematic representation of the rorf3-grlRA region. The bent arrow indicates the grlRA transcriptional start site determined in this study. The binding regions for H-NS and Ler revealed by EMSAs are represented by hatched and open boxes, respectively. The DNA fragments used in EMSAs are represented below the diagram of the rorf3-grlRA region. The sizes of the fragments are indicated to the left, and the corresponding fusion numbers are indicated to the right. Increasing concentrations of purified Ler-His6 (B) or H-NS-His6 (C) protein were incubated with the PCR-generated DNA fragments represented in panel A, resolved in 4% polyacrylamide gels, and stained with ethidium bromide. The fragments correspond to the pCRglrRA transcriptional fusions, as indicated to the left of the gels. The EPEC ler structural gene was used as a negative control (NC) for the EMSAs. Arrows indicate DNA-protein complexes.
FIG. 7.
FIG. 7.
Model for the regulation of LEE genes in A/E pathogens. Ler positively regulates LEE gene expression by counteracting the H-NS-mediated repression of LEE gene promoters. The expression of ler is tightly regulated by specific regulators, such as GrlA, GrlR, and PerC, as well as by global regulators, such as IHF, Fis, BipA, EtrA, EivF, and QseA. Appropriate levels of ler expression are maintained by a positive regulatory loop formed by Ler and GrlA, which could be negatively modulated by GrlR through a mechanism that is still not well understood or by the ability of Ler to negatively autoregulate its own expression (see the text).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology. John Wiley and Sons, New York, N.Y.
    1. Berdichevsky, T., D. Friedberg, C. Nadler, A. Rokney, A. Oppenheim, and I. Rosenshine. 2005. Ler is a negative autoregulator of the LEE1 operon in enteropathogenic Escherichia coli. J. Bacteriol. 187:349-357. - PMC - PubMed
    1. Buchet, A., W. Nasser, K. Eichler, and M. A. Mandrand-Berthelot. 1999. Positive co-regulation of the Escherichia coli carnitine pathway cai and fix operons by CRP and the CaiF activator. Mol. Microbiol. 34:562-575. - PubMed
    1. Bustamante, V. H., J. A. Ibarra, K. Carrillo, A. Vazquez, and J. L. Puente. 2004. The locus of enterocyte effacement-encoded regulator (Ler) overcomes repression of type III secretion operons by modifying a nucleoprotein complex formed by H-NS. Presented at the 104th General Meeting of the American Society for Microbiology, New Orleans, La., 23 to 27 May 2004.
    1. Bustamante, V. H., F. J. Santana, E. Calva, and J. L. Puente. 2001. Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression. Mol. Microbiol. 39:664-678. - PubMed

Publication types

LinkOut - more resources

-