Modelling fim expression in Escherichia coli K12

Fimbriae are structures in Escherichia coli, the expression of which is controlled by the fim operon. Understanding this expression is important because the fimbriae are important virulence factors. This expression can be studied using targeted mutations to the DNA, which can be used to disable binding or transcription of a protein. How- ever, this can be problematic as only the net effect is observed. Turning off expression of a protein may enhance fim expression, but deactivating this protein may also repress another protein that functions as an activator of fim expression. The net result may be that fim expression goes down, so it would seem at first glance that the disabled protein was an activator of fim expression and not a repressor. In order to understand this complex network of interactions, an agent based model of fim expression has been created. The subject of this paper is to introduce this model and to use it to disambiguate between a number of hypotheses about this system. Parameters such as binding probability will be optimised using a genetic algorithm. The final model and parameters show a good match to experimental data

Multicellular Computing Using Conjugation for Wiring

Recent efforts in synthetic biology have focussed on the implementation of logical functions within living cells. One aim is to facilitate both internal ‘‘re-programming’’ and external control of cells, with potential applications in a wide range of domains. However, fundamental limitations on the degree to which single cells may be re-engineered have led to a growth of interest in multicellular systems, in which a ‘‘computation’’ is distributed over a number of different cell types, in a manner analogous to modern computer networks. Within this model, individual cell type perform specific sub-tasks, the results of which are then communicated to other cell types for further processing. The manner in which outputs are communicated is therefore of great significance to the overall success of such a scheme. Previous experiments in distributed cellular computation have used global communication schemes, such as quorum sensing (QS), to implement the ‘‘wiring’’ between cell types. While useful, this method lacks specificity, and limits the amount of information that may be transferred at any one time. We propose an alternative scheme, based on specific cell-cell conjugation. This mechanism allows for the direct transfer of genetic information between bacteria, via circular DNA strands known as plasmids. We design a multicellular population that is able to compute, in a distributed fashion, a Boolean XOR function. Through this, we describe a general scheme for distributed logic that works by mixing different strains in a single population; this constitutes an important advantage of our novel approach. Importantly, the amount of genetic information exchanged through conjugation is significantly higher than the amount possible through QS-based communication. We provide full computational modelling and simulation results, using deterministic, stochastic and spatially-explicit methods. These simulations explore the behaviour of one possible conjugation-wired cellular computing system under different conditions, and provide baseline information for future laboratory implementations

Adaptation and diversification of <i>Escherichia coli</i> K12 MC1000 in a complex environment

Complexiteit is inherent aan natuurlijke zowel als industriele habitats. Voorgaand wetenschappelijk werk heeft duidelijk het flexibele (genotypische en fenotypische) aanpassingsvermogen van microorganismen aan complexiteit laten zien. De meeste experimenten zijn echter onder relatief simpele (uniforme) omstandigheden verricht. Derhalve richtte het huidige onderzoek zich op bacteriele evolutie in complex groeimedium, waarbij de nadruk lag op de analyse van de mate van genetische / fysiologische diversifiering naar fitnessverhoging en nichedifferentiatie.De lange-termijn-aanpassingen (~1000 generaties) van E. coli K12 MC1000 in Luria-Bertani (LB) bouillon onder aerobe, wisselende en anaerobe condities werden geevalueerd. Verschillende genetische wegen resulteerden in aanpassingen en een aantal metabole routes waren geactiveerd. De veranderingen waren reproduceerbaar met betrekking tot geselecteerde functie, waarbij habitat de belangrijkste selector bleek. Een specific response werd waargenomen in de genen die betrokken waren bij het metabolisme van galactose (galR en galE). Daarbij werd een hoge mate van heterogeniteit gevonden tussen en binnen populaties. De verschillende fenotypische aanpassingen gaven ook aan dat parallele responses werden gestuurd door de verschillende genomen.De analyse van polymorfismen binnen een geevolueerde population toonde het bestaan van twee metabole and interactieve typen aan. Derhalve werd het voorkomen van additionele specifieke fenotypische eigenschappen (stress resistentie en metabole eigenschappen) bevestigd. De interactieve en stabiele coexistentie van deze vormen liet trade-offs in groei- en stress-eigenschappen tussen de vormen, en nicheverdeling, zien. De complexiteit van de habitat kan derhalve de vorming van aangepaste coexisterende vormen sturen.An inherent characteristic of natural as well as industrial environments is complexity. Scientific studies have revealed the flexible genetic and phenotypic capacities of microorganisms to cope with such complexity. However, most experiments have been conceptually simple, as they compare populations adapting to rather uniform environments. Therefore, the present work addressed bacterial evolution in a complex environment. The emphasis was on unraveling the level of diversification in respect of the genetic and physiological changes that the organism underwent, which allowed it to either acquire superior fitness or occupy a different niche.The long-term (~1000 generations) adaptive responses of E. coli K12 MC1000 in Luria-Bertani (LB) broth under aerobic, fluctuating and anaerobic conditions were evaluated. Several genetic solutions led to adaptation and a number of metabolic pathways were activated. Reproducibility of changes on genuine targets of selection was observed in parallel populations, suggesting a response triggered by medium. A specific response occurred in genes related to the metabolisms of galactose (galR and galE). Considerable heterogeneity was also found between and within populations. Differential phenotypic outcomes, suggested that parallel responses were affected by differing genomic backgrounds. Analysis of the polymorphisms in one evolved population revealed the existence of two main metabolic and interactive types. The emergence of additional specific phenotypic traits (stress resistance and metabolic properties) was confirmed. The interactive and stable coexistence of these forms revealed the presence of trade-offs and niche partitioning. The complexity of the environment has the potential to trigger the establishment of adapted and coexisting forms

Doctor of Philosophy

dissertationProkaryotes make extensive use of posttranscriptional regulation to modulate diverse cellular processes such as central carbon metabolism, stress response pathways, and virulence determinants. Posttranscriptional regulation in Escherichia coli is mediated via two broadly characterized methods. The first utilizes small noncoding RNAs (sRNAs) which bind target mRNA transcripts to alter their stability and translation. Nearly all characterized sRNAs function jointly with an RNA chaperone protein, Hfq. The second method employs mRNA-binding proteins which directly mediate translational inhibition or activation upon mRNA targets. Posttranscriptional regulation by both methods was recently demonstrated important to pathogenesis by several bacterial organisms. This study addresses the role of posttranscriptional regulation in uropathogenic Escherichia coli (UPEC), the organisms responsible for the majority of urinary tract infections. Specifically, deletion of Hfq, an RNA chaperone required for many sRNA-mRNA interactions, strongly reduced infection in murine models of cystitis and pyelonephritis and virtually eliminated formation of UPEC intracellular bacterial communities (IBCs). The hfq mutant experienced severe sensitivities to membrane disrupting agents such as polymyxin B, reactive oxygen species (ROS) and reactive nitrogen species (RNS) during in vitro models of host innate immune function. These phenotypes mirrored those of a !E-deleted UPEC, suggesting Hfq's involvement in posttranscriptional regulation of virulence was largely exerted at the bacterial envelope. In addition, RNS-treatment of ! ! "#! UPEC resulted in posttranscriptional downregulation of CpxP, a periplasmic regulator of the Cpx envelope stress response pathway. This downregulation was dependent on carbon storage regulator A (CsrA), a protein posttranscriptional regulator, as overexpression of CsrB, an sRNA antagonist of CsrA function, was sufficient to prevent as well as overcome downregulation of CpxP by RNS. Overexpression of CpxP in the presence of RNS proved beneficial to growth, however, suggesting CpxP downregulation by urinary RNS may not just disrupt UPEC's envelope, but impair the Cpx pathway involved in its repair. Anti-nitrotyrosine immunoblotting and mass-spectrometry indicate nitrosation of CsrA at tyrosine 48, a residue immediately adjacent to the domain implicated in RNA interaction, possibly altering CsrA's binding properties. These results demonstrate posttranscriptional regulation assisting virulence, but also imply manipulation by the host to deter growth

Molecular determinants of surface colonisation in diarrhoeagenic Escherichia coli (DEC): from bacterial adhesion to biofilm formation

peer-reviewedEscherichia coli is primarily known as a commensal colonising the gastrointestinal tract of infants very early in life but some strains being responsible for diarrhoea, which can be especially severe in young children. Intestinal pathogenic E. coli include six pathotypes of diarrhoeagenic E. coli (DEC), namely, the (i) enterotoxigenic E. coli, (ii) enteroaggregative E. coli, (iii) enteropathogenic E. coli, (iv) enterohemorragic E. coli, (v) enteroinvasive E. coli and (vi) diffusely adherent E. coli. Prior to human infection, DEC can be found in natural environments, animal reservoirs, food processing environments and contaminated food matrices. From an ecophysiological point of view, DEC thus deal with very different biotopes and biocoenoses all along the food chain. In this context, this review focuses on the wide range of surface molecular determinants acting as surface colonisation factors (SCFs) in DEC. In the first instance, SCFs can be broadly discriminated into (i) extracellular polysaccharides, (ii) extracellular DNA and (iii) surface proteins. Surface proteins constitute the most diverse group of SCFs broadly discriminated into (i) monomeric SCFs, such as autotransporter (AT) adhesins, inverted ATs, heat-resistant agglutinins or some moonlighting proteins, (ii) oligomeric SCFs, namely, the trimeric ATs and (iii) supramolecular SCFs, including flagella and numerous pili, e.g. the injectisome, type 4 pili, curli chaperone-usher pili or conjugative pili. This review also details the gene regulatory network of these numerous SCFs at the various stages as it occurs from pre-transcriptional to post-translocational levels, which remains to be fully elucidated in many cases