SummaryEnteropathogenic Escherichia coli (EPEC) elicit changes in host cell morphology and cause actin rearrangement, a phenotype that has commonly been referred to as attaching/effacing (AE) lesions. The ability of EPEC to induce AE lesions is dependent upon a type III protein secretion/translocation system that is encoded by genes clustered in a 35.6 kb DNA segment, named the locus of enterocyte effacement (LEE). We used transcriptional fusions between the green fluorescent protein (gfp) reporter gene and LEE genes rorf2, orf3, orf5, escJ, escV and eae, together with immunoblot analysis with antibodies against Tir, intimin, EspB and EspF, to analyse the genetic regulation of the LEE. The expression of all these LEE genes was strictly dependent upon the presence of a functional integration host factor (IHF). IHF binds specifically upstream from the ler (orf1) promoter and appears to activate expression of ler, orf3, orf5 and rorf2 directly. The ler-encoded Ler protein was involved in activating the expression of escJ, escV, tir, eae, espB and espF. Expression of both IHF and Ler was needed to elicit actin rearrangement associated with AE lesions. In conclusion, IHF directly activates the expression of the ler and rorf2 transcriptional units, and Ler in turn mediates the expression of the other LEE genes.

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhoea in young children. The locus of enterocyte effacement (LEE) pathogenicity island comprises a cluster of operons encoding a type III secretion system and related proteins that are associated with EPEC virulence. The LEE1 operon encodes Ler that positively regulates the LEE2, LEE3, LEE4, LEE5 and espG transcriptional units. The LEE operons are repressed at 27 SC and expressed at 37 SC. This paper describes a regulatory cascade of the thermoregulation of LEE operons. LEE1 including ler is repressed by H-NS at 27 SC but not at 37 SC. In contrast, the expression of the LEE2, LEE3, LEE4, LEE5 and espG transcriptional units is repressed by H-NS at both 27 SC and 37 SC. Upon shifting the culture temperature from 27 SC to 37 SC, Ler is synthesized and in turn activates the expression of LEE2, LEE3, LEE4 and espG by releasing the H-NS mediated repression. In the case of LEE5, Ler acts both by alleviating the H-NS mediated repression and by an additional mechanism, as yet to be defined.

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (TTSS) to inject effector proteins into the plasma membrane and cytosol of infected cells. To translocate proteins, EPEC, like Salmonella and Shigella, is believed to assemble a macromolecular complex (type III secreton) that spans both bacterial membranes and has a short needle‐like projection. However, there is a special interest in studying the EPEC TTSS owing to the fact that one of the secreted proteins, EspA, is assembled into a unique filamentous structure also required for protein translocation. In this report we present electron micrographs of EspA filaments which reveal a regular segmented substructure. Recently we have shown that deletion of the putative structural needle protein, EscF, abolished protein secretion and formation of EspA filaments. Moreover, we demonstrated that EspA can bind directly to EscF, suggesting that EspA filaments are physically linked to the EPEC needle complex. In this paper we provide direct evidence for the association between an EPEC bacterial membrane needle complex and EspA filaments, defining a new class of filamentous TTSS.

Lrp (leucine-responsive regulatory protein) is a major Escherichia coli regulatory protein which regulates expression of a number of operons, some negatively and some positively. Operons that are affected by the presence or absence of Lrp includefanABC (3), gcv (10), ginA (5), gltBD (5), ilvIH (20), livJ and livK (7), lysU (6, 11), ompC and ompF (5), oppABCDF (1), papBA (3), sdaA (12), serA (12, 21), and tdh (12, 21). Some of these operons (e.g., ilvIH) are known to be directly controlled by Lrp, whereas others (e.g., ginA) are affected only indirectly by Lrp. Other operons that are affected by Lrp were identified by a plac Mu mutational analysis and by two-dimensional gel electrophoresis (5, 10). Many operons under control of Lrp are also subject to control by leucine. A striking feature of the Lrp regulon is the variety of ways that leucine and Lrp interact to regulate gene expression (16). Of the operons activated by Lrp, in some cases the activation requires leucine, in some cases the activation is negated by leucine, and in other cases the activation is independent of leucine. Similarly, for operons which are negatively regulated by Lrp, the same three subcategories have been observed: leucine negates the effect, leucine is required for the effect, and leucine has no effect. The molecular mechanisms underlying these six different patterns of regulation involving Lrp and leucine are only partially understood.Lrp, a dimer containing two identical subunits of molecular mass 18.8 kDa, is present in E. coli at a level of about 3,000 molecules per cell (32). Its amino acid sequence is evolutionarily related to that of AsnC, a regulatory protein that controls

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Enteropathogenic Escherichia coli (EPEC) are extracellular pathogens that colonize mucosal surfaces of the intestine via formation of attaching and effacing (A/E) lesions. The genes responsible for induction of the A/E lesions are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes the adhesin intimin and the type III secretion system needle complex, translocator and effector proteins. One of the major EPEC translocator proteins, EspA, forms a filamentous conduit along which secreted proteins travel before they arrive at the translocation pore in the plasma membrane of the host cell, which is composed of EspB and EspD. Prior to secretion, many type III proteins, including translocators, are maintained in the bacterial cytoplasm by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins Tir, Map and EspF, and the translocator proteins EspD and EspB. In this study, CesAB (Orf3 of the LEE) was identified as a chaperone for EspA and EspB. Specific CesAB–EspA and CesAB–EspB protein interactions are demonstrated. CesAB was essential for stability of EspA within the bacterial cell prior to secretion. Furthermore, a cesAB mutant failed to secrete EspA, as well as EspB, to assemble EspA filaments, to induce A/E lesion following infection of HEp-2 cells and to adhere to, or cause haemolysis of, erythrocytes.

Transformation of the high-CO2-requiring mutants (hcr) 0221 and El derived from the cyanobacterium Synechococcus sp. strain PCC 7942 by a wild-type DNA library restored their ability to grow at the level of CO2 in air. A plasmid (pE12) containing a 10-kilobase DNA insert was rescued from a 0221 heterogenote and proved to transform both 0221 and El to the wild-type phenotype. The capacity of the pE12 subclones to confer the wild-type phenotype to 0221 transformants enabled the mapping of the mutation in 0221 (designated hcrO221) within a 232-base-pair PstI-BstXI DNA restriction fragment. Sequence analysis revealed two open reading frames (ORFs) at positions -1745 to -1262 (ORFI) and -1218 to -393 (ORFiH) upstream of the rbcL gene. A 3-kilobase PstI fragment of 0221 was cloned, and hcrO221 was found to be a point mutation within the PstI-BstXI region -1309 nucleotides upstream of the rbcL gene. The significance of this flanking region for adaptation to air levels of CO2 was further demonstrated by the generation of new hcr mutants following insertional inactivation of wild-type DNA in the BstXI site. Electron microscopy revealed aberrant carboxysome structures in growing cells of the hcr mutants, a defect that was possibly related to the mutation, since transformation with pE12 derivatives restored the carboxysome structure to normal.The photosynthetic performance of photosynthetic organisms is limited by the availability of CO2 in air to ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase because of the low affinity of the carboxylase for CO2 and the competition between CO2 and 02 for the reaction with RuBP (2, 3, 6). Cyanobacteria and green algae possess an inducible mechanism for concentrating inorganic carbon (C,) internally to levels high enough for efficient carboxylation (1,3,4,(11)(12)(13). This mechanism enables them to adapt to the level of CO2 in air (0.03%) and to grow photoautotrophically in this environment (12,15). During the adaptation process, the photosynthetic affinity to external Ci (3,12,16), the rate of C, transport (3, 12, 15, 17), and the synthesis of a 42-kilodalton cytoplasmic membrane protein (19,20) are increased. Furthermore, cyanobacteria growing at the air level of CO2 contain more carboxysomes than those growing at 5% CO2 in air do (30). In both cyanobacteria and photosynthetic bacteria, these bodies contain a significant amount of RuBP carboxylase (5, 6). It has been suggested that this subcellular location of the enzyme is important for the efficient utilization of CO2 (22).In an attempt to elucidate the molecular basis of the adaptation process, we have isolated mutants from Synechococcus sp. strain PCC 7942 that failed to adapt to the air level of CO2 (15, 25) but that grew normally at 5% CO2 in air. In these mutants the utilization of the internal Ci pool for photosynthesis is altered, while the accumulation of C, appears to be normal (15,21,25). Molecular analysis of these mutants is a valuable tool for identifying the putative gene(s) and product(s) involved in the adaptat...

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. Essential for colonization of the host intestine is the LEE pathogenicity island, which comprises a cluster of operons encoding a type III secretion system and related proteins. The LEE1 operon encodes Ler, which positively regulates many EPEC virulence genes in the LEE region and elsewhere in the chromosome. We found that Ler acts as a specific autorepressor of LEE1 transcription. We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro. A comparison of the Ler affinities to different DNA regions suggests that the autoregulation mechanism limits the steady-state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance maximizing the colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the autoregulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression. Our findings point to a new negative regulatory circuit that suppresses the noise and optimizes the expression levels of ler and other LEE1 genes.Colonizing enteropathogens compete with the gut flora to gain a foothold in the host tissue by expressing powerful colonization factors. However, to reduce the immune response of the host, the pathogen should minimize the expression of the colonization factors. To resolve this dilemma, pathogens evolved regulatory mechanisms that optimize the expression levels and timing, thus maintaining expression of just enough colonization factors and only when needed. Another layer of complexity is added when the colonization is dependent on the assembly of organelles like the type III secretion systems (TTSS), which are composed of ϳ30 different proteins of various relative amounts and encoded by several operons. In these cases, an orderly expression program is required for efficient assembly of the organelle.Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. It employs the TTSS as a molecular syringe to inject a battery of toxic or colonization proteins into the membrane and cytoplasm of infected host cells (4). The TTSS and some of the effectors are encoded by a 35.6-kbp pathogenicity island, termed the locus for enterocyte effacement (LEE). The LEE consists of 41 genes, organized in five major operons (LEE1 to LEE5) and several additional transcriptional units (10,19). Ler, an H-NS paralog, encoded by the first gene of the LEE1 operon, is a key regulator of the LEE regulon, positively regulating expression of LEE2, LEE3, LEE4, LEE5, espG, and map (11,19,24,30). The regulation of ler (LEE1 operon) is complex and involves many factors, including H-NS, integration host factor (IHF), Fis, PerC, BipA, GrlA, GrlR, GadX, and quorum sensing (2,7,11,13,14,17,19,26,28,30,32). Most of these factors appe...