Vanessa Sperandio

Quorum sensing Escherichia coli regulators B and C (QseBC): A novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli

Quorum sensing is a cell‐to‐cell signalling mechanism in which bacteria secrete hormone‐like compounds called autoinducers. When these auto‐inducers reach a certain threshold concentration, they interact with bacterial transcriptional regulators, thereby regulating gene expression. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 as well as E. coli K‐12 produces the autoinducer‐2 (AI‐2), which is synthesized by the product of the luxS gene, and previous work from our laboratory has shown that genes encoding the EHEC type III secretion system were activated by quorum sensing. Recently, by hybridizing an E. coli K‐12 gene array with cDNA synthesized from RNA extracted from EHEC strain 86‐24 and its isogenic luxS mutant, we observed that other potential virulence‐associated factors, such as genes encoding the expression and assembly of flagella, motility and chemotaxis, were also activated by quorum sensing. The array data also indicated that several genes encoding putative E. coli regulators were controlled by quorum sensing. In this report, we describe a two‐component system regulated by quorum sensing that shares homology with Salmonella typhimurium PmrAB, which we have named quorum sensing E. coli regulator B and C (QseBC). The qseBC genes, previously identified only as open reading frames b3025 and b3026, are organized in an operon in the E. coli chromosome, with qseB encoding the response regulator and qseC the sensor kinase. We confirmed the regulation of qseBC by quorum sensing using qseB::lacZ transcriptional fusions and characterized the phenotypes of an isogenic qseC mutation in EHEC. This mutant expressed less flagellin and had reduced motility compared with the wild‐type and complemented strains. Transcription of flhD, fliA, motA and fliC::lacZ fusions was decreased in the qseC mutant, suggesting that qseBC is a transcriptional regulator of flagella genes. A qseC mutant was also generated in E. coli K‐12 strain MC1000 that showed the same phenotypes as the EHEC mutant, indicating that qseBC regulates flagella and motility by quorum sensing in both EHEC and K‐12. QseBC activates transcription of flhDC, which is the master regulator for the flagella and motility genes and, in the absence of flhD, QseBC failed to activate the transcription of fliA. Motility of a luxS, but not of a qseC, mutant can be restored by providing AI‐2 exogenously as preconditioned media, suggesting that the qseC mutant is unable to respond to AI‐2. However, QseC has no effect on the expression of other quorum sensing‐controlled genes such as those encoding for the type III secretion system. These data indicate that QseBC is one component of the quorum‐sensing regulatory cascade in both EHEC and K‐12 that is involved in the regulation of flagella and motility genes, but that additional regulators in this cascade remain to be characterized.

Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 microm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with lambda-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

The Locus of Enterocyte Effacement (LEE)-Encoded Regulator Controls Expression of Both LEE- and Non-LEE-Encoded Virulence Factors in Enteropathogenic and Enterohemorrhagic Escherichia coli

ABSTRACT Regulation of virulence gene expression in enteropathogenicEscherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2, LEE3, andLEE4 promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2, orf10,rorf10, orf19, and espF, indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. Δler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.

Quorum-sensing Escherichia coli regulator A : a regulator of the LysR family involved in the regulation of the locus of enterocyte effacement pathogenicity island in enterohemorrhagic E. coli

ABSTRACT The locus of enterocyte effacement (LEE) is a chromosomal pathogenicity island that encodes the proteins involved in the formation of the attaching and effacing lesions by enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). The LEE comprises 41 open reading frames organized in five major operons, LEE1, LEE2, LEE3, tir (LEE5), and LEE4, which encode a type III secretion system, the intimin adhesin, the translocated intimin receptor (Tir), and other effector proteins. The first gene of LEE1 encodes the Ler regulator, which activates all the other genes within the LEE. We previously reported that the LEE genes were activated by quorum sensing through Ler (V. Sperandio, J. L. Mellies, W. Nguyen, S. Shin, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 96:15196-15201, 1999). In this study we report that a putative regulator in the E. coli genome is itself activated by quorum sensing. This regulator is encoded by open reading frame b3243; belongs to the LysR family of regulators; is present in EHEC, EPEC, and E. coli K-12; and shares homology with the AphB and PtxR regulators of Vibrio cholerae and Pseudomonas aeruginosa, respectively. We confirmed the activation of b3243 by quorum sensing by using transcriptional fusions and renamed this regulator quorum-sensing E. coli regulator A (QseA). We observed that QseA activated transcription of ler and therefore of the other LEE genes. An EHEC qseA mutant had a striking reduction of type III secretion activity, which was complemented when qseA was provided in trans. Similar results were also observed with a qseA mutant of EPEC. The QseA regulator is part of the regulatory cascade that regulates EHEC and EPEC virulence genes by quorum sensing.

Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler.

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells. The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir. The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE‐encoded regulator (Ler). The LEE2 and LEE3 operons are divergently transcribed with overlapping −10 promoter regions, and gene fusion studies have shown that they are both activated by Ler. Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than −247u2003bp lead to loss of activation by Ler, whereas only 70u2003bp upstream of the LEE3 transcription start site is required for Ler‐mediated activation. We have purified Ler as a His‐tagged protein and used it to perform DNA‐binding assays with LEE2 and LEE3. We observed that Ler bound to a DNA fragment containing the −300 to +1 region of LEE2; however, it failed to bind to a DNA fragment containing the −300 to +1 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2. The Ler‐activatable LEE3::lacZ fusions extended to what would be −246u2003bp of the LEE2 operon. A lacZ fusion from the −300 to +1 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site. DNase I footprinting revealed that Ler protected a region of 121u2003bp upstream of LEE2. Purified Ler mutated in the coiled‐coil domain was unable to activate transcription and to bind to the LEE2 regulatory region. These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure.