Environment Controls LEE Regulation in Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a significant cause of infant morbidity and mortality in developing regions of the world. Horizontally acquired genetic elements encode virulence structures, effectors, and regulators that promote bacterial colonization and disease. One such genetic element, the locus of enterocyte effacement (LEE), encodes the type three secretion system (T3SS) which acts as a bridge between bacterial and host cells to pass effector molecules that exert changes on the host. Due to its importance in EPEC virulence, regulation of the LEE has been of high priority and its investigation has elucidated many virulence regulators, including master regulator of the LEE Ler, H-NS, other nucleoid-associated proteins, GrlA, and PerC. Media type, environmental signals, sRNA signaling, metabolic processes, and stress responses have profound, strain-specific effects on regulators and LEE expression, and thus T3SS formation. Here we review virulence gene regulation in EPEC, which includes approaches for lessening disease by exploiting the elucidated regulatory pathways

Enteropathogenic and Enterohemorrhagic Escherichia coli Virulence Gene Regulation▿

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Interaction of Ler at the LEE5 (tir) Operon of Enteropathogenic Escherichia coli

The genome of enteropathogenic Escherichia coli (EPEC) encodes a global regulator, Ler (locus of enterocyte effacement [LEE]-encoded regulator), which activates expression of several polycistronic operons within the 35.6-kb LEE pathogenicity island, including the LEE2-LEE3 divergent operon pair containing overlapping −10 regions and the LEE5 (tir) operon. Ler is a predicted 15-kDa protein that exhibits amino acid similarity with the nucleoid protein H-NS. In order to study Ler-mediated activation of virulence operons in EPEC, we used a molecular approach to characterize the interactions of purified Ler protein with the upstream regulatory sequences of the LEE5 operon. We determined the cis-acting DNA sequences necessary for Ler binding at LEE5 by mobility shift and DNase I protection assays, demonstrating that Ler acts directly at LEE5 by binding sequences between positions −190 and −73 in relation to the transcriptional start site. Based on the molecular weight of Ler, the similarity to H-NS, and the extended region of protection observed in a DNase I footprint at LEE5, we hypothesized that multiple Ler proteins bind upstream of the LEE5 promoter to increase transcriptional activity from a distance. Using an hns deletion strain, we demonstrated that like the LEE2-LEE3 operon pair, H-NS represses LEE5 transcription. We describe a model in which Ler activates transcription at both divergent overlapping paired and single promoters by displacing H-NS, which results in the disruption of a repressing nucleoprotein complex

The Global Regulator Ler Is Necessary for Enteropathogenic Escherichia coli Colonization of Caenorhabditis elegans

Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries and is useful for general investigations of the bacterial infection process. However, the study of the molecular pathogenesis of EPEC has been hampered by the lack of genetically tractable, convenient animal models. We have therefore developed the use of the nematode Caenorhabditis elegans as a small animal model of infection for this diarrheal pathogen. We found that nematodes died faster on nematode growth medium in the presence of EPEC pathogens than in the presence of the laboratory control strain MG1655. Increased numbers of pathogens in the gut, determined by standard plate count assays and fluorescence microscopy using green fluorescent protein-expressing bacteria, correlated with killing. Deletion of the gene encoding the global regulator Ler severely reduced the ability of EPEC to colonize the nematode gut and could be complemented by providing the ler gene on a multicopy plasmid in trans. Neither the type III secretion system nor the type IV bundle-forming pilus was required for colonization. Combined, the similarities and distinct differences between EPEC infection of nematodes and that of humans offer a unique opportunity to study several stages of the infection process, namely, attachment, colonization, and persistence, in a genetically tractable, inexpensive, and convenient in vivo system

Comparative sequence analysis of the plasmid-encoded regulator of enteropathogenic Escherichia coli Strains

Enteropathogenic Escherichia coli (EPEC) strains that carry the EPEC adherence factor (EAF) plasmid were screened for the presence of different EAF sequences, including those of the plasmid-encoded regulator (per). Considerable variation in gene content of EAF plasmids from different strains was seen. However, bfpA, the gene encoding the structural subunit for the bundle-forming pilus, bundlin, and per genes were found in 96.8% of strains. Sequence analysis of the per operon and its promoter region from 15 representative strains revealed that it is highly conserved. Most of the variation occurs in the 5′ two-thirds of the perA gene. In contrast, the C-terminal portion of the predicted PerA protein that contains the DNA-binding helix-turn-helix motif is 100% conserved in all strains that possess a full-length gene. In a minority of strains including the O119:H2 and canine isolates and in a subset of O128:H2 and O142:H6 strains, frameshift mutations in perA leading to premature truncation and consequent inactivation of the gene were identified. Cloned perA, -B, and -C genes from these strains, unlike those from strains with a functional operon, failed to activate the LEE1 operon and bfpA transcriptional fusions or to complement a per mutant in reference strain E2348/69. Furthermore, O119, O128, and canine strains that carry inactive per operons were deficient in virulence protein expression. The context in which the perABC operon occurs on the EAF plasmid varies. The sequence upstream of the per promoter region in EPEC reference strains E2348/69 and B171-8 was present in strains belonging to most serogroups. In a subset of O119:H2, O128:H2, and O142:H6 strains and in the canine isolate, this sequence was replaced by an IS1294-homologous sequence

Microbial Consortia and Mixed Plastic Waste: Pangenomic Analysis Reveals Potential for Degradation of Multiple Plastic Types via Previously Identified PET Degrading Bacteria

The global utilization of single-use, non-biodegradable plastics, such as bottles made of polyethylene terephthalate (PET), has contributed to catastrophic levels of plastic pollution. Fortunately, microbial communities are adapting to assimilate plastic waste. Previously, our work showed a full consortium of five bacteria capable of synergistically degrading PET. Using omics approaches, we identified the key genes implicated in PET degradation within the consortium’s pangenome and transcriptome. This analysis led to the discovery of a novel PETase, EstB, which has been observed to hydrolyze the oligomer BHET and the polymer PET. Besides the genes implicated in PET degradation, many other biodegradation genes were discovered. Over 200 plastic and plasticizer degradation-related genes were discovered through the Plastic Microbial Biodegradation Database (PMBD). Diverse carbon source utilization was observed by a microbial community-based assay, which, paired with an abundant number of plastic- and plasticizer-degrading enzymes, indicates a promising possibility for mixed plastic degradation. Using RNAseq differential analysis, several genes were predicted to be involved in PET degradation, including aldehyde dehydrogenases and several classes of hydrolases. Active transcription of PET monomer metabolism was also observed, including the generation of polyhydroxyalkanoate (PHA)/polyhydroxybutyrate (PHB) biopolymers. These results present an exciting opportunity for the bio-recycling of mixed plastic waste with upcycling potential