Development of novel orthogonal genetic circuits, based on extracytoplasmic function (ECF) σ factors

The synthetic biology field aims to apply the engineering 'design-build-test-learn' cycle for the implementation of synthetic genetic circuits modifying the behavior of biological systems. In order to reach this goal, synthetic biology projects use a set of fully characterized biological parts that subsequently are assembled into complex synthetic circuits following a rational, model-driven design. However, even though the bottom-up design approach represents an optimal starting point to assay the behavior of the synthetic circuits under defined conditions, the rational design of such circuits is often restricted by the limited number of available DNA building blocks. These usually consist only of a handful of transcriptional regulators that additionally are often borrowed from natural biological systems. This, in turn, can lead to cross-reactions between the synthetic circuit and the host cell and eventually to loss of the original circuit function. Thus, one of the challenges in synthetic biology is to design synthetic circuits that perform the designated functions with minor cross-reactions (orthogonality). To overcome the restrictions of the widely used transcriptional regulators, this project aims to apply extracytoplasmic function (ECF) σ factors in the design novel orthogonal synthetic circuits. ECFs are the smallest and simplest alternative σ factors that recognize highly specific promoters. ECFs represent one of the most important mechanisms of signal transduction in bacteria, indeed, their activity is often controlled by anti-σ factors. Even though it was shown that the overexpression of heterologous anti-σ factors can generate an adverse effect on cell growth, they represent an attractive solution to control ECF activity. Finally, to date, we know thousands of ECF σ factors, widespread among different bacterial phyla, that are identifiable together with the cognate promoters and anti-σ factors, using bioinformatic approaches. All the above-mentioned features make ECF σ factors optimal candidates as core orthogonal regulators for the design of novel synthetic circuits. In this project, in order to establish ECF σ factors as standard building blocks in the synthetic biology field, we first established a high throughput experimental setup. This relies on microplate reader experiments performed using a highly sensitive luminescent reporter system. Luminescent reporters have a superior signal-to-noise ratio when compared to fluorescent reporters since they do not suffer from the high auto-fluorescence background of the bacterial cell. However, they also have a drawback represented by the constant light emission that can generate undesired cross-talk between neighboring wells on a microplate. To overcome this limitation, we developed a computational algorithm that corrects for luminescence bleed-through and estimates the “true” luminescence activity for each well of a microplate. We show that the correcting algorithm preserves low-level signals close to the background and that it is universally applicable to different experimental conditions. In order to simplify the assembly of large ECF-based synthetic circuits, we designed an ECF toolbox in E. coli. The toolbox allows for the combinatorial assembly of circuits into expression vectors, using a library of reusable genetic parts. Moreover, it also offers the possibility of integrating the newly generated synthetic circuits into four different phage attachment (att) sites present in the genome of E. coli. This allows for a flawless transition between plasmid-encoded and chromosomally integrated genetic circuits, expanding the possible genetic configurations of a given synthetic construct. Moreover, our results demonstrate that the four att sites are orthogonal in terms of the gene expression levels of the synthetic circuits. With the purpose of rationally design ECF-based synthetic circuits and taking advantage of the ECF toolbox, we characterized the dynamic behavior of a set of 15 ECF σ factors, their cognate promoters, and relative anti-σs. Overall, we found that ECFs are non-toxic and functional and that they display different binding affinities for the cognate target promoters. Moreover, our results show that it is possible to optimize the output dynamic range of the ECF-based switches by changing the copy number of the ECFs and target promoters, thus, tuning the input/output signal ratio. Next, by combining up to three ECF-switches, we generated a set of “genetic-timer circuits”, the first synthetic circuits harboring more than one ECF. ECF-based timer circuits sequentially activate a series of target genes with increasing time delays, moreover, the behavior of the circuits can be predicted by a set of mathematical models. In order to improve the dynamic response of the ECF-based constructs, we introduced anti-σ factors in our synthetic circuits. By doing so we first confirmed that anti-σ factors can exert an adverse effect on the growth of E. coli, thus we explored possible solutions. Our results demonstrate that anti-σ factors toxicity can be partially alleviated by generating truncated, soluble variants of the anti-σ factors and, eventually, completely abolished via chromosomal integration of the anti-σ factor-based circuits. Finally, after demonstrating that anti-σ factors can be used to generate a tunable time delay among ECF expression and target promoter activation, we designed ECF/AS-suicide circuits. Such circuits allow for the time-delayed cell-death of E. coli and will serve as a prototype for the further development of ECF/AS-based lysis circuits

Resolving stalled replication forks in Escherichia coli

DNA replication is essential to successful cell proliferation. Inheritance of traits during cell propagation relies on the accurate duplication of the parental double-stranded DNA (dsDNA) to form two identical daughter copies. This process is carried out by a multi-protein complex referred to as the replisome. Decades of investigations using the model Escherichia coli (E. coli) replisome have provided an overall picture of the process of DNA replication initiation, elongation and termination. However, DNA replication in cells occurs on template DNA coated in DNA-binding proteins that can act as roadblocks and stall the replisome, often resulting in drastic effects on the chromosome. However, the fate of the replisome at these sites remains poorly understood. Stalled DNA replication has been linked to the emergence of antimicrobial resistance in prokaryotes, and the development of severe physical disorders and diseases in eukaryotes. Therefore, understanding the underlying mechanisms of stalled DNA replication can inform future investigations into the maintenance of genome integrity. This thesis focuses on the development and use of single-molecule tools to investigate stalled replication and the resolution of protein roadblocks. Single-molecule tools provide the ability to watch one molecule at a time. Extensive use of these techniques has revealed the heterogeneity that exists within complex biological pathways. Specifically, this thesis highlights the myriad of previously unknown behaviors of proteins on DNA as revealed by single-molecule tools

Replication arrest and bypass at Tus/ter complexes in the terminus of the Escherichia coli chromosome

Genome replication is frequently challenged by obstacles that can result from DNA damage, topological stress or tightly bound proteins. Replication fork stalling at DNA-bound proteins can lead to collapse of the fork and promote mutation and genomic instability, a hallmark of cancer cells. Interaction between replisomes and naturally occurring barriers can provide important information for understanding genome instability mechanisms. The simplicity of the Escherichia coli chromosome replication is ideal for studies of complex interactions between replication forks and replication barriers. E. coli carries a single chromosome that encodes a single origin of bidirectional replication, oriC, and a region diametrically opposite to oriC where replication terminates. The terminus region encodes four 23 bp ter sites, terA and terD on right replichore and terC and terB on the left. A ter sequence bound by Tus protein acts as a polar (unidirectional) natural barrier to fork progression. The Tus/ter system allows replisomes to enter the terminus, but will arrest their progress into the opposite replichore, that is, towards oriC. In this work two dimensional native-native gel electrophoresis was utilized to detect stalled replication forks at naturally occurring Tus/ter barriers in the E. coli chromosome terminus. The majority of arrested replication forks were found to accumulate at the first Tus/ter barrier on the left replichore, Tus/terC. Notably fewer arrested forks were detected at terA, terB and terD. The strength of ter sites was shown to be independent of the location in the terminus, whereas the sequence of ter sites was critical. The terB sequence forms the strongest terminator and restricts frequent replication fork bypass observed at terC. This correlates with the published data on the strength of nucleoprotein barriers formed by ter sequences observed in vitro. The presence of a strong terminator on each replichore helps the Tus/ter system prevent unwanted replication to escape the terminus. In the situation where additional rounds of replication were initiated in the terminus in the absence of the RecG helicase, most of replication forks were able to bypass Tus/terC barrier and were arrested at Tus/terB. Previous studies, in the Michel laboratory, revealed that the UvrD helicase can promote the bypass of a synthetically introduced Tus/terB replication fork barrier in the middle of the right replichore, but only as a consequence of RecA-mediated homologous recombination. The work presented in this thesis shows that, even in the absence of RecA-mediated homologous recombination, UvrD could promote the bypass of the naturally occurring Tus/terC (soft) barrier in the chromosome terminus. However, UvrD was unable to promote fork movement through stronger Tus/terB and Tus/terA barriers in the terminus. The terC and terB nucleotide sequences differ in three separate segments. I have shown that, one of the segments outside of the conserved region plays a critical role in the UvrD-dependent replication fork bypass of the Tus/terC barrier. My results suggest a distinct role of the UvrD helicase in the alleviation of replication fork stalling at the naturally occurring Tus/terC barrier in the chromosomal terminus

When push comes to shove - RNA polymerase and DNA-bound protein roadblocks

In recent years, transcriptional roadblocking has emerged as a crucial regulatory mechanism in gene expression, whereby other DNA-bound obstacles can block the progression of transcribing RNA polymerase (RNAP), leading to RNAP pausing and ultimately dissociation from the DNA template. In this review, we discuss the mechanisms by which transcriptional roadblocks can impede RNAP progression, as well as how RNAP can overcome these obstacles to continue transcription. We examine different DNA-binding proteins involved in transcriptional roadblocking and their biophysical properties that determine their effectiveness in blocking RNAP progression. The catalytically dead CRISPR-Cas (dCas) protein is used as an example of an engineered programmable roadblock, and the current literature in understanding the polarity of dCas roadblocking is also discussed. Finally, we delve into a stochastic model of transcriptional roadblocking and highlight the importance of transcription factor binding kinetics and its resistance to dislodgement by an elongating RNAP in determining the strength of a roadblock.Nan Hao, Alana J. Donnelly, Ian B. Dodd, Keith E. Shearwi

Development of novel orthogonal genetic circuits, based on extracytoplasmic function (ECF) σ factors

The synthetic biology field aims to apply the engineering 'design-build-test-learn' cycle for the implementation of synthetic genetic circuits modifying the behavior of biological systems. In order to reach this goal, synthetic biology projects use a set of fully characterized biological parts that subsequently are assembled into complex synthetic circuits following a rational, model-driven design. However, even though the bottom-up design approach represents an optimal starting point to assay the behavior of the synthetic circuits under defined conditions, the rational design of such circuits is often restricted by the limited number of available DNA building blocks. These usually consist only of a handful of transcriptional regulators that additionally are often borrowed from natural biological systems. This, in turn, can lead to cross-reactions between the synthetic circuit and the host cell and eventually to loss of the original circuit function. Thus, one of the challenges in synthetic biology is to design synthetic circuits that perform the designated functions with minor cross-reactions (orthogonality). To overcome the restrictions of the widely used transcriptional regulators, this project aims to apply extracytoplasmic function (ECF) σ factors in the design novel orthogonal synthetic circuits. ECFs are the smallest and simplest alternative σ factors that recognize highly specific promoters. ECFs represent one of the most important mechanisms of signal transduction in bacteria, indeed, their activity is often controlled by anti-σ factors. Even though it was shown that the overexpression of heterologous anti-σ factors can generate an adverse effect on cell growth, they represent an attractive solution to control ECF activity. Finally, to date, we know thousands of ECF σ factors, widespread among different bacterial phyla, that are identifiable together with the cognate promoters and anti-σ factors, using bioinformatic approaches. All the above-mentioned features make ECF σ factors optimal candidates as core orthogonal regulators for the design of novel synthetic circuits. In this project, in order to establish ECF σ factors as standard building blocks in the synthetic biology field, we first established a high throughput experimental setup. This relies on microplate reader experiments performed using a highly sensitive luminescent reporter system. Luminescent reporters have a superior signal-to-noise ratio when compared to fluorescent reporters since they do not suffer from the high auto-fluorescence background of the bacterial cell. However, they also have a drawback represented by the constant light emission that can generate undesired cross-talk between neighboring wells on a microplate. To overcome this limitation, we developed a computational algorithm that corrects for luminescence bleed-through and estimates the “true” luminescence activity for each well of a microplate. We show that the correcting algorithm preserves low-level signals close to the background and that it is universally applicable to different experimental conditions. In order to simplify the assembly of large ECF-based synthetic circuits, we designed an ECF toolbox in E. coli. The toolbox allows for the combinatorial assembly of circuits into expression vectors, using a library of reusable genetic parts. Moreover, it also offers the possibility of integrating the newly generated synthetic circuits into four different phage attachment (att) sites present in the genome of E. coli. This allows for a flawless transition between plasmid-encoded and chromosomally integrated genetic circuits, expanding the possible genetic configurations of a given synthetic construct. Moreover, our results demonstrate that the four att sites are orthogonal in terms of the gene expression levels of the synthetic circuits. With the purpose of rationally design ECF-based synthetic circuits and taking advantage of the ECF toolbox, we characterized the dynamic behavior of a set of 15 ECF σ factors, their cognate promoters, and relative anti-σs. Overall, we found that ECFs are non-toxic and functional and that they display different binding affinities for the cognate target promoters. Moreover, our results show that it is possible to optimize the output dynamic range of the ECF-based switches by changing the copy number of the ECFs and target promoters, thus, tuning the input/output signal ratio. Next, by combining up to three ECF-switches, we generated a set of “genetic-timer circuits”, the first synthetic circuits harboring more than one ECF. ECF-based timer circuits sequentially activate a series of target genes with increasing time delays, moreover, the behavior of the circuits can be predicted by a set of mathematical models. In order to improve the dynamic response of the ECF-based constructs, we introduced anti-σ factors in our synthetic circuits. By doing so we first confirmed that anti-σ factors can exert an adverse effect on the growth of E. coli, thus we explored possible solutions. Our results demonstrate that anti-σ factors toxicity can be partially alleviated by generating truncated, soluble variants of the anti-σ factors and, eventually, completely abolished via chromosomal integration of the anti-σ factor-based circuits. Finally, after demonstrating that anti-σ factors can be used to generate a tunable time delay among ECF expression and target promoter activation, we designed ECF/AS-suicide circuits. Such circuits allow for the time-delayed cell-death of E. coli and will serve as a prototype for the further development of ECF/AS-based lysis circuits

DNA replication and replication termination in Escherichia coli

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonA prerequisite for successful cell division is the generation of an accurate copy of the entire genome as well as faithful segregation into the daughter cells. In the bacterium Escherichia coli, replication of the circular chromosome is initiated at a single origin (oriC) where two replication forks are assembled and proceed bi-directionally until they converge within a defined termination region opposite oriC and fork fusion takes place. This region is flanked by ter sequences, which, when bound by Tus protein, form a replication fork trap that allows forks to enter but not to leave. While the events associated with initiation as well as the elongation of replication have been extensively studied, the molecular details associated with the fusion of two replisomes are far less well characterised. The data presented here significantly extend our understanding of the molecular mechanics associated with the fusion of two replisomes in E. coli. My results strongly support the idea that RecG is a key player in processing intermediates that arise as two replication forks fuse. In the absence of RecG, over-replication of the chromosome is initiated at fork fusion intermediates, a process that can take place outside of the native termination area if forks are forced to fuse in an ectopic location. RecG has also been implicated in processing recombination intermediates. Over-replication in the absence of RecG is dependent on recombination and my data support the idea that RecG is important in limiting replication that initiates at recombination intermediates, some of which arise as a consequence of fork fusion events. In contrast, my data do not support the notion that the over-replication of chromosomal DNA in cells lacking RecG is in any way triggered by R-loops, as suggested previously in the literature. The observation that over-replication is taking place in cells lacking exonuclease I strongly supports the idea that 3’ single-stranded DNA structures are a key intermediate of fork fusion events. My data demonstrate that 3’ flaps accumulating in the absence of ExoI can be converted into 5’ flaps and degraded by 5’ exonucleases such as ExoVII and RecJ. RecG helicase is likely to be involved in this conversion. Thus, the data presented in this thesis highlight the complexity of replication fork fusion events and demonstrate that multiple protein activities are required to process fork fusion intermediates in order to allow DNA replication to be completed with high accuracy and enable faithful segregation of complete chromosomes into daughter cells

Obstacles to DNA replication in Escherichia coli and the role of UvrD helicase in their resolution

UvrD is a multi-functional Escherichia coli helicase. It is widely involved in DNA repair, including its DNA unwinding role during mismatch repair and RNAP removal role during transcription coupled repair. UvrD has also been shown to colocalise with the replisome during DNA replication and has an important role in nucleoprotein block removal to clear the path for the replication fork. The main aims of this project were to test UvrD ability to unwind DNA and displace nucleoprotein blocks and to test whether addition of the mismatch protein MutL could improve UvrD function. Our in vitro results showed that UvrD alone was capable of unwinding past a physiologically relevant block, Tus-terB, in ‘easy’ and ‘challenging’ conditions. The addition of MutL increased the ability of UvrD to unwind long double-stranded DNA substrates in multiple challenging conditions as well as against a physiologically unfamiliar nucleoprotein block (EcoRI E111G). Our results showed that UvrD activity appeared to be enhanced when tasked with unwinding past the Tus-terB block and presented better DNA unwinding alone rather than with MutL. To understand more about the UvrD:MutL physical and functional interaction, we designed two UvrD mutants with large N-terminal deletions for UvrD:MutL interaction tests. Additionally, since UvrD:MutL complex formation causes the rotation of the UvrD 2B subdomain, we designed a UvrD∆2B mutant, to test for MutL interaction and determine how the 2B subdomain deletion affects UvrD function. We also developed a new in vitro biochemical assay designed to test for the displacement of Tus by the replisome during DNA replication termination. Our preliminary findings suggest that replication forks colliding with the non-permissive orientation of the Tus-ter block can displace Tus. Establishment of the Tus jumping assay will allow us to investigate the interaction of DNA replication forks with Tus-ter and to test whether other proteins, such as UvrD, are involved in replication termination

Replication Fork Breakage and Restart in Escherichia coli

In all organisms, replication impairments are an important source of genome rearrangements, mainly because of the formation of double-stranded DNA (dsDNA) ends at inactivated replication forks. Three reactions for the formation of dsDNA ends at replication forks were originally described for Escherichia coli and became seminal models for all organisms: the encounter of replication forks with preexisting single-stranded DNA (ssDNA) interruptions, replication fork reversal, and head-to-tail collisions of successive replication rounds. Here, we first review the experimental evidence that now allows us to know when, where, and how these three different reactions occur in E. coli. Next, we recall our recent studies showing that in wild-type E. coli, spontaneous replication fork breakage occurs in 18% of cells at each generation. We propose that it results from the replication of preexisting nicks or gaps, since it does not involve replication fork reversal or head-to-tail fork collisions. In the recB mutant, deficient for double-strand break (DSB) repair, fork breakage triggers DSBs in the chromosome terminus during cell division, a reaction that is heritable for several generations. Finally, we recapitulate several observations suggesting that restart from intact inactivated replication forks and restart from recombination intermediates require different sets of enzymatic activities. The finding that 18% of cells suffer replication fork breakage suggests that DNA remains intact at most inactivated forks. Similarly, only 18% of cells need the helicase loader for replication restart, which leads us to speculate that the replicative helicase remains on DNA at intact inactivated replication forks and is reactivated by the replication restart proteins

Investigation of Mobile Genetic Elements and Antimicrobial Resistance Genes in Human Oral Metagenomic DNA

Antibiotic resistance is currently one of the major global healthcare problems. Bacteria can become resistant by acquiring resistance genes from other bacteria. This process is usually facilitated by mobile genetic elements (MGEs), a type of DNA that can move from one site to another site within bacterial genome, and often between bacterial cells. The human oral cavity has been shown to harbour various antimicrobial resistance genes (ARGs). The aim of this research is to study the fundamental biology and the association between MGEs and ARGs present in human oral bacteria by both sequence and functional-based metagenomic assays. Using a PCR-based method, various genes predicted to confer antimicrobial resistance and other adaptive traits were identified on different MGEs (composite transposons, integrons and novel MGEs called translocatable units). This is the first report that showed ARGs in the human oral cavity were associated with these MGEs, especially in integron gene cassettes (GCs). Some of the integron gene cassettes were predicted to not contain any genes at all. They were predicted to have a regulatory function as a promoter, which could be important for the expression of other genes carried by integrons. Using an enzymatic reporter assay, it was proven that one of the functions of these GCs is as a promoter, which could allow bacteria to survive multiple stresses within the complex environment of the human oral cavity. Functional screening of a metagenomic library identified a clone that can confer resistance to two commonly used antiseptics agents. This was shown to be a result of UDP-glucose 4-epimerase enzyme derived from a common oral bacteria Veillonella parvula, which altered the cell’s surface charge to be more positive, presumably reducing the binding of positively charges antiseptics to the bacteria. To tackle the antibiotic resistance problems effectively, the understanding of the nature of MGEs is crucial. We have shown the presence of multiple novel MGEs, ARGs and a novel resistance mechanism. Those detected ARGs can be used for the surveillance and increase the understanding of MGEs in other environments

Defining the ancestral replication fork trap in Tus-dependent bacteria

Casey Toft used in silico and in vitro approaches to refine the fork trap architecture in Tus-dependent bacteria and its potential application in Immuno-PCR diagnostics. He discovered a ubiquitous 'ancestral' replication fork trap architecture consisting of only two Terminator sites at odds with the well-established and complex Escherichia coli system