April 21, 2020  |  

The Complete Genome of the Atypical Enteropathogenic Escherichia coli Archetype Isolate E110019 Highlights a Role for Plasmids in Dissemination of the Type III Secreted Effector EspT.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Genome and Functional Characterization of Colonization Factor Antigen I- and CS6-Encoding Heat-Stable Enterotoxin-Only Enterotoxigenic Escherichia coli Reveals Lineage and Geographic Variation

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of childhood diarrhea and is a leading cause of travelertextquoterights diarrhea. ETEC strains encoding the heat-stable enterotoxin (ST) are more often associated with childhood diarrhea than ETEC strains that encode only the heat-labile enterotoxin (LT). Colonization factors (CFs) also have a demonstrated role in ETEC virulence, and two of the most prevalent CFs among ETEC that have caused diarrhea are colonization factor antigen I (CFA/I) and CS6. In the current report, we describe the genomes of 269 CS6- or CFA/I-encoding ST-only ETEC isolates that were associated with human diarrhea. While the CS6 and CFA/I ETEC were identified in at least 13 different ETEC genomic lineages, a majority (85%; 229/269) were identified in only six lineages. Complete genome sequencing of selected isolates demonstrated that a conserved plasmid contributed to the dissemination of CFA/I whereas at least five distinct plasmids were involved in the dissemination of ST and/or CS6. Additionally, there were differences in gene content between CFA/I and CS6 ETEC at the phylogroup and lineage levels and in association with their geographic location of isolation as well as lineage-related differences in ST production. Thus, we demonstrate that genomically diverse E. coli strains have acquired ST, as well as CFA/I or CS6, via one or more plasmids and that, in some cases, isolates of a particular lineage or geographic location have undergone additional modifications to their genome content. These findings will aid investigations of virulence and the development of improved diagnostics and vaccines against this important human diarrheal pathogen.IMPORTANCE Comparative genomics and functional characterization were used to analyze a global collection of CFA/I and CS6 ST-only ETEC isolates associated with human diarrhea, demonstrating differences in the genomic content of CFA/I and CS6 isolates related to CF type, lineage, and geographic location of isolation and also lineage-related differences in ST production. Complete genome sequencing of selected CFA/I and CS6 isolates enabled descriptions of a highly conserved ST-positive (ST+) CFA/I plasmid and of at least five diverse ST and/or CS6 plasmids among the CS6 ETEC isolates. There is currently no approved vaccine for ST-only ETEC, or for any ETEC for that matter, and as such, the current report provides functional verification of ST and CF production and antimicrobial susceptibility testing and an in-depth genomic characterization of a collection of isolates that could serve as representatives of CFA/I- or CS6-encoding ST-only ETEC strains for future studies of ETEC pathogenesis, vaccine studies, and/or clinical trials.


April 21, 2020  |  

Deciphering bacterial epigenomes using modern sequencing technologies.

Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression, virulence and pathogen-host interactions.


April 21, 2020  |  

Genome-wide systematic identification of methyltransferase recognition and modification patterns.

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.


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