Matthew D. Johnson

A systems biology approach sheds new light on Escherichia coli acid resistance

In order to develop an infection, diarrhogenic Escherichia coli has to pass through the stomach, where the pH can be as low as 1. Mechanisms that enable E. coli to survive in low pH are thus potentially relevant for pathogenicity. Four acid response systems involved in reducing the concentration of intracellular protons have been identified so far. However, it is still unclear to what extent the regulation of other important cellular functions may be required for survival in acid conditions. Here, we have combined molecular and phenotypic analysis of wild-type and mutant strains with computational network inference to identify molecular pathways underlying E. coli response to mild and strong acid conditions. The interpretative model we have developed led to the hypothesis that a complex transcriptional programme, dependent on the two-component system regulator OmpR and involving a switch between aerobic and anaerobic metabolism, may be key for survival. Experimental validation has shown that the OmpR is responsible for controlling a sizeable component of the transcriptional programme to acid exposure. Moreover, we found that a ΔompR strain was unable to mount any transcriptional response to acid exposure and had one of the strongest acid sensitive phenotype observed.

Novel aspects of the acid response network of E. coli K-12 are revealed by a study of transcriptional dynamics.

Understanding gene regulation and its adaptive significance requires not only a detailed knowledge of individual molecular interactions that give rise to changes in gene expression but also an overview of complete genetic networks and the ways in which components within them interact. Increasingly, such studies are being done using luminescent or fluorescent reporter proteins that enable monitoring of gene expression dynamics in real time, particularly during changes in expression. We show here that such an approach is valid for dissecting the responses of the AR2 or GAD network of Escherichia coli K-12 to changes in pH, which is one of the most complex networks known in E. coli. In addition to confirming several regulatory interactions that have been revealed by previous studies, this approach has identified new components in this system that lead to complex dynamics of gene expression following a drop in pH, including an auto-regulatory loop involving the YdeO activator protein and novel roles for the PhoP protein.

Laboratory adapted Escherichia coli K-12 becomes a pathogen of Caenorhabditis elegans upon restoration of O antigen biosynthesis

Escherichia coli has been the leading model organism for many decades. It is a fundamental player in modern biology, facilitating the molecular biology revolution of the last century. The acceptance of E. coli as model organism is predicated primarily on the study of one E. coli lineage; E. coli K‐12. However, the antecedents of todays laboratory strains have undergone extensive mutagenesis to create genetically tractable offspring but which resulted in loss of several genetic traits such as O antigen expression. Here we have repaired the wbbL locus, restoring the ability of E. coli K‐12 strain MG1655 to express the O antigen. We demonstrate that O antigen production results in drastic alterations of many phenotypes and the density of the O antigen is critical for the observed phenotypes. Importantly, O antigen production enables laboratory strains of E. coli to enter the gut of the Caenorhabditis elegans worm and to kill C. elegans at rates similar to pathogenic bacterial species. We demonstrate C. elegans killing is a feature of other commensal E. coli. We show killing is associated with bacterial resistance to mechanical shear and persistence in the C. elegans gut. These results suggest C. elegans is not an effective model of human‐pathogenic E. coli infectious disease.

RcsB Is Required for Inducible Acid Resistance in Escherichia coli and Acts at gadE-Dependent and -Independent Promoters

RcsB interacts with GadE to mediate acid resistance in stationary-phase Escherichia coli K-12. We show here that RcsB is also required for inducible acid resistance in exponential phase and that it acts on promoters that are not GadE regulated. It is also required for acid resistance in E. coli O157:H7.

Transcription of the plasmid-encoded toxin gene from Enteroaggregative Escherichia coli is regulated by a novel co-activation mechanism involving CRP and Fis

Enteroaggregative Escherichia coli (EAEC) is a major cause of diarrhoea in developing countries. EAEC 042 is the prototypical strain. EAEC 042 secretes the functionally well‐characterized Pet autotransporter toxin that contributes to virulence through its cytotoxic effects on intestinal epithelial cells. Following a global transposon mutagenesis screen of EAEC 042, the transcription factors, CRP and Fis, were identified as essential for transcription of the pet gene. Using both in vivo and in vitro techniques, we show that the pet promoter is co‐dependent on CRP and Fis. We present a novel co‐activation mechanism whereby CRP is placed at a non‐optimal position for transcription initiation, creating dependence on Fis for full activation of pet. This study complements previous findings that establish Fis as a key virulence regulator in EAEC 042.

Characterization of mutations in the PAS domain of the EvgS sensor kinase selected by laboratory evolution for acid resistance in Escherichia coli

Laboratory‐based evolution and whole‐genome sequencing can link genotype and phenotype. We used evolution of acid resistance in exponential phase Escherichia coli to study resistance to a lethal stress. Iterative selection at pH 2.5 generated five populations that were resistant to low pH in early exponential phase. Genome sequencing revealed multiple mutations, but the only gene mutated in all strains was evgS, part of a two‐component system that has already been implicated in acid resistance. All these mutations were in the cytoplasmic PAS domain of EvgS, and were shown to be solely responsible for the resistant phenotype, causing strong upregulation at neutral pH of genes normally induced by low pH. Resistance to pH 2.5 in these strains did not require the transporter GadC, or the sigma factor RpoS. We found that EvgS‐dependent constitutive acid resistance to pH 2.5 was retained in the absence of the regulators GadE or YdeO, but was lost if the oxidoreductase YdeP was also absent. A deletion in the periplasmic domain of EvgS abolished the response to low pH, but not the activity of the constitutive mutants. On the basis of these results we propose a model for how EvgS may become activated by low pH.

Vinyl glycosides in oligosaccharide synthesis (part 5): A latent-active glycosylation strategy for the preparation of branched trisaccharide libraries

Abstract For the first time, an efficient strategy is described for the preparation of orthogonally protected branched and linear saccharide libraries which can be further functionalised. A small number of key building blocks can be converted into a wide range of glycosyl donors and acceptors.

Visualising Multiple Data Sources in an Independent Open Learner Model

This paper introduces the Next-TELL independent open learner model which is constructed based on data from a range of sources. An example is presented for a university course, with the learner model built from the main activities undertaken during the course. Use of the Next-TELL open learner model over a five week period is described for this group of students, suggesting that independent open learner models built from multiple sources of data may have much to offer in supporting students’ understanding of their learning, and could potentially be used to encourage greater peer interaction.

Structural basis of subtilase cytotoxin subAB assembly

Background: AB5 toxins consist of a pentameric B-subunit and a catalytic A-subunit. Results: Crystallographic data, dissociation, and intracellular trafficking of SubAB toxin are reported. Conclusion: SubAB architecture is similar to other AB5 toxins, whereas the B-pentamer plays an important role in assembly and intracellular trafficking. Significance: The conserved hydrophobic ring in the B-pentamer supports the view that A- and B-subunits have evolved independently. Pathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhance or inhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin.

Design of a Computer-assisted Assessment System for classroom formative assessment

We describe a number of high-level design decisions that we found essential for a Computer-assisted Assessment System that is to be deployed in school classrooms for supporting formative assessment by teachers and self-assessment by students. In addition, the system needs to provide information to parents. Our design decisions comprise the use of the Open Learner Model approach to make diagnostic information available to the various stakeholders, the use of a modelling methodology to describe assessment methods declaratively (glass-box), and the decision to embed assessment in a flexible manner into current and emerging learning environments. Implications for system architecture are also described.