A MATHEMATICAL MODEL TO STUDY THE ROLE OF THE LSR INTERGENIC REGION IN MEDIATION OF AUTOINDUCER-2 QUORUM SENSING IN ESCHERICHIA COLI

Quorum sensing (QS) is a process that allows bacteria to communicate with each other to coordinate collective behavior in response to changes in environmental conditions. Their ability to mediate biofilm formation of biofilms and antibiotic resistance has created challenges on healthcare systems, and an impetus for us to understand QS systems. QS mediated by autoinducer-2 is likely to be the most common of these mechanisms. Recent work has elaborated on the LuxS-regulated (Lsr) system which can mediate and process AI-2 to QS-dependent behaviors, particularly regulatory elements including the lsr intergenic region and the repressor LsrR, the so-called QS"switch". In this thesis, we present a simulation of an example lsr-QS-system to elucidate the role of the lsr intergenic region binding site interactions and how this model integrates with recent literature on LsrR's protein structure to provide further details on the mechanisms of how the switch may operate in real systems

LOCAL AND GLOBAL GENE REGULATION ANALYSIS OF THE AUTOINDUCER-2 MEDIATED QUORUM SENSING MECHANISM IN ESCHERICHIA COLI

The term `quorum sensing' (QS) is used to define a population density based communication mechanism which uses chemical signal molecules called autoinducers to trigger unique and varied changes in gene expression. Although several communication methods have been identified in bacteria that are unique to a particular species, one type of signal molecule, autoinducer-2 (AI-2) is linked to interspecies communication, indicating its potential as a universal signal for cueing a QS response among multiple bacterial types. In E. coli, AI-2 acts as an effector by binding to the QS repressor LsrR. As a result, LsrR unbinds and relieves repression of the lsr regulon, stimulating a subsequent QS gene expression cascade. In this dissertation, LsrR structure and in vitro binding activity are examined. Genomic binding and DNA microarray analyses are conducted and three novel sites putatively regulated by LsrR, yegE-udk, mppA and yihF, are revealed. Two cAMP receptor protein (CRP) binding locations in intergenic region of the lsr regulon are also confirmed. The role of each CRP site in divergent expression is qualified, indicating the lsr intergenic region to be a class III CRP-dependent promoter. Also, four specific DNA binding sites for LsrR in the lsr intergenic region are proposed, and reliance upon simultaneous binding to these various sites and the resulting effects on LsrR repression is presented. Finally, a complex model for regulation of the lsr regulon is depicted incorporating LsrR, CRP, DNA looping, and a predicted secondary layer of repression by an integration host factor (IHF)-like protein. Further understanding of this QS genetic mechanism may potentially be used for inhibiting bacterial proliferation and infection, modifying the natural genetic system to elicit alternate desired responses, or extracted and applied to a highly customizable and sensitive in vitro biosensor

Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits

Because cells have the extraordinary ability to sense and respond to even subtle environmental changes by intricately regulating their gene expression patterns, their behaviors can be intentionally “tuned” by altering the state of their environments in a prescribed or rational manner. Rational control of both external and internal molecular stimuli provides a basis for many biotechnological applications including the expression of foreign protein products. This is done by coordinately controlling product synthesis while retaining the cell in a productive state. Quorum sensing (QS), a molecular signaling modality that mediates cell-cell communication, autonomously facilitates both inter- and intra-species gene regulation. This process can be rewired to enable autonomously actuated, but molecularly programmed, genetic control. Recently, even electrical signals, which have long been used to control the most sophisticated of man-made devices, are now employed to alter cell signaling processes enabling computer programmed behavior, particularly in cells suitably engineered to accommodate electrical signals. By minimally engineering these genetic circuits, new applications have emerged for the repurposing of Escherichia coli, from creating innovative sensor concepts to stimulating the emerging field of electrogenetics

SYSTEMATIC INVESTIGATION OF QUORUM SENSING IN Escherichia coli

High throughput techniques and advanced mathematical tools have enabled systematic investigations of biological systems with unparalleled precision. Not only molecular interactions between components but mechanisms and the dynamic behaviors associated with these systems are revealed, suggesting that comprehensive systems biology can be realized in the near future. Quorum sensing, especially the auto-inducer2 (AI-2) system, has been extensively studied due to its commonality among bacteria and connections to pathogenic phenotypes. In this study, the E. coli quorum sensing AI-2 system was studied combing system-based mathematical modeling and high throughput genomic profiling. First, a Stochastic Petri Network (SPN) model was constructed based on available regulatory information. Simulations together with experimental data demonstrated that the apparent stimulation of AI-2 in the presence of glucose is not from the increased transcriptional or translational expression of AI-2 synthases luxS and pfs, nor from the increased metabolic flux associated with LuxS-related pathways but from an alternative AI-2 synthesis pathway. The conversion of adenosine with cellular extracts from both luxS and pfs mutants validated our prediction about the existence of an alternative non-LuxS related AI-2 synthesis pathway. Second, AI-2 uptake regulatory network was investigated in detail: lsrR-lacZ, lsrK-lacZ fusion reporters were constructed and the analysis found that lsrR is subject to its own repression and is induced by both lsrK and luxS. Further transcriptome analysis demonstrated that lsrR and lsrK, together with quorum signal AI-2, coregulate lsrRK regulon, which influences phenotypes (biofilm, small RNAs). Importantly, this regulation is in a distinctly different manner than that mediating the lsr operon. We hypothesize that lsrR acts together with AI-2 to mediate cellular processes and that the phosphorylation of AI-2 molecule through lsrK triggers different response pathways. These investigations demonstrated that lsrR, lsrK are indispensable for AI-2 uptake. These newly elucidated regulatory mechanisms and associations undoubtedly broaden the scope of the AI-2 quorum sensing system, and provide a solid foundation for further mathematical modeling of the dynamics and system behaviors in E. coli . Finally, a tight coupling of experimental manipulation with mathematical analysis, as demonstrated in this study, provides a good example for systematically investigating biological systems

CONTEXTUALIZATION OF THE E. COLI LSR SYSTEM: RELATIVE ORTHOLOGY, RELATIVE QS ACTIVITY, AND EMERGENT BEHAVIOR

Within bacterial consortia there exist innumerable combinatorial circumstances, some of which may tip the scale toward pathogenicity, some of which may favor asymptomatic phenotypes. Indeed, the lines and intersections between commensal, pathogenic, and opportunistic bacteria are not always clean. As a foothold to mediate pathogenicity arising from consortia, many have puzzled at communication between bacteria. Primary among such considerations is quorum sensing (QS). Analogous to autocrine signaling in multicellular organisms, QS is a self-signaling process involving small molecules. Generally, QS activation is believed to have pleiotropic effects, and has been associated with numerous pathogenic phenotypes. The research herein focuses on autoinducer-2 (AI-2) based QS signaling transduced through the Lsr system. Produced by over 80 species of bacteria, AI-2 is believed to be an interspecies signaling molecule. Outside of the marine bacteria genera Vibrio and Marinomonas, the only known AI-2 based QS transduction pathway is the Lsr system. We sought to deepen the characterization of the Lsr system in contexts outside of the batch cultures in which it was originally defined. First, we interrogated E. coli K-12 W3110 Lsr system orthologs relative to the same strain's lac system. Both systems are induced by the molecule which they import and catabolize. We searched for homologs by focusing on the gene order along a genome, as gene arrangement can bear signaling consequences for autoregulatory circuits. We found that the Lsr system signal was phylogenetically dispersed if not particularly deep, especially outside of Enterobacteriales and Pasteurellaceaes, indicating that the system has generally been conferred horizontally. This contrasts with the lac system, whose signal is strong but limited to a select group of highly related enterobacteria. We then modeled the Lsr system with ODEs, revealing bimodality in silico, bolstering preliminary experimental evidence. This bifurcated expression was seen to depend upon nongenetic heterogeneity, which we modeled as a variation of a single compound parameter, basal, representing the basal rate of AI-2 flux into the cell through a low flux pathway. Moreover, in our finite difference-agent based models, bimodal expression could not arise from spatial stochasticity alone. This lies in contrast with the canonical LuxIR QS system, which employs an intercellular positive feedback loop to activate the entire population. We examined the consequences of this contrast, by modeling both systems under conditions of colony growth using finite difference-agent based methods. We additionally investigated the confluence of Lsr signaling with chemotactic sensitivity to AI-2, which has been demonstrated in E. coli. Finally, the consequences of bimodality in interspecies interactions were assessed by posing two populations containing different Lsr systems against each other. While few natural consortia consist of only two interacting bacteria, these studies indicate that AI-2 based Lsr signaling may mediate a multitude of transitional intraspecies and interspecies bacterial dynamics, the specifics of which will vary with the context and the homologs involved

PROGRAMMING BACTERIAL CONSORTIA FOR AUTONOMOUS REGULATION AND COORDINATED ACTIVITY

The potential of genetically engineered microbes seems nearly infinite with applications ranging from human health to bioprocessing. However, metabolic burden and unbalanced use of cell resources are frequent challenges when engineering cells to carry out synthetic functions. To work around this challenge, engineers are attempting to use co-cultures or synthetic consortia wherein labor is divided amongst subpopulations that work together. This emerging strategy requires new tools to regulate the composition of subpopulations and to enable robust coordination between subpopulations. Here, we investigated and rewired a native cell-cell communication process, quorum sensing, in order to develop tools to regulate co-cultures. We developed modules for signal regulated cell growth rate and cell-cell communication in bacteria, and we used these modules to construct co-cultures with autonomous composition control. Specifically, we developed a “controller” strain for signal modulated cell growth rate by using quorum sensing signals to regulate levels of HPr, a protein involved in sugar transport. We developed a second “translator” strain that detects the universal quorum sensing signal AI-2 and translates it into a species-specific AI-1 signal. The composition of the resulting co-culture adjusts autonomously in response to AI-2. Importantly, we developed a simple mathematical model based on individual monocultures that predicts behavior of the co-culture. Then, we used our model to explore in silico alternate construct designs operating in varied environments. To complement the co-culture model, which explores behavior due to interactions between strains but does not encompass information about the genetic circuits underlying the quorum sensing process, we then developed a gene circuit model of a dual-input synthetic AI-2 quorum sensing system. Finally, we demonstrate that the strategies developed in our co-culture platform can be used to engineer co-cultures where the culture composition is controlled electrically. We also show that these strategies can be used to change the culture composition of a synthetic co-culture where each population is working together to produce pyocyanin, thereby changing the rate of pyocyanin production in the co-culture. The techniques developed here may enable further use of co-cultures or synthetic consortia by synthetic biologists and metabolic engineers for varied applications

Novel function and regulation of mutagenic DNA polymerases in Escherichia coli

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2007.Vita.Includes bibliographical references.The observation that mutations in the Escherichia coli genes umuC+ and umuD+ abolish mutagenesis induced by UV-light strongly supported the counterintuitive notion that such mutagenesis is an active rather than passive process. Biochemical studies have revealed that umuC+ and its homolog dinB+ encode novel, low to moderate fidelity DNA polymerases with the ability to catalyze synthesis on imperfect DNA templates in a process termed translesion synthesis (TLS). Similar enzymes exist in nearly all organisms, constituting the Y-superfamily of DNA polymerases. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, its precise function has remained elusive. Here we show that AdinB E. coli strains are sensitive to DNA damaging agents that form lesions at the N2 position of guanine. In vitro bypass studies of an N2-guanine adduct by DinB demonstrate considerable preference for correct nucleotide insertion and an increased catalytic proficiency on the lesion-bearing template relative to undamaged DNA. Moreover, DinB and its mammalian and archaeal orthologs possess similar substrate specificities. Mutation of a single residue in the active site ofE. coli DinB suggests that its enhanced activity is coupled to lesion recognition and that its TLS function is required for resistance to DNA damaging agents in vivo.(cont.) Regulation of the mutagenic potential of DinB is critical for maintenance of genomic integrity. We present evidence indicating that abortive TLS products generated by a DinB variant are subject to the proofreading function of DNA polymerase III. Moreover, both the TLS activity and -1 frameshift mutator potential of DinB are modulated in a highly sophisticated manner by the DNA damage-inducible proteins RecA and UmuD2. These biochemical data, coupled with genetic analyses and molecular modeling, indicate that DinB is a specialized and remarkably controlled translesion DNA polymerase. In addition, we present evidence that the umuC+participates in several novel biological functions in addition to its established role in TLS. A novel umuC gain-of-function allele confers striking resistance to hydroxyurea and umuC+ mediates the expression of genes and physiological responses under conditions of SOS induction. Taken together, these observations hint at at a largely uncharacterized function of Y-family polymerases in sculpting physiological responses, including active mechanisms of cell death, in response to environmental stress.by Daniel F. Jarosz.Ph.D