Coronatine-insensitive 1 (COI1) mediates transcriptional responses of <i>Arabidopsis thaliana</i> to external potassium supply

The ability to adjust growth and development to the availability of mineral nutrients in the soil is an essential life skill of plants but the underlying signaling pathways are poorly understood. In &lt;i&gt;Arabidopsis thaliana&lt;/i&gt;, shortage of potassium (K) induces a number of genes related to the phytohormone jasmonic acid (JA). Using comparative microarray analysis of wild-type and coi1-16 mutant plants, we classified transcriptional responses to K with respect to their dependence on COI1, a central component of oxylipin signaling. Expression profiles obtained in a short-term experiment clearly distinguished between COI1-dependent and COI1-independent K-responsive genes, and identified both known and novel targets of JA-COI1-signaling. During long-term K-deficiency, coi-16 mutants displayed de novo responses covering similar functions as COI1-targets except for defense. A putative role of JA for enhancing the defense potential of K-deficient plants was further supported by the observation that plants grown on low K were less damaged by thrips than plants grown with sufficient K

A novel parametric approach to mine gene regulatory relationship from microarray datasets

<p>Abstract</p> <p>Background</p> <p>Microarray has been widely used to measure the gene expression level on the genome scale in the current decade. Many algorithms have been developed to reconstruct gene regulatory networks based on microarray data. Unfortunately, most of these models and algorithms focus on global properties of the expression of genes in regulatory networks. And few of them are able to offer intuitive parameters. We wonder whether some simple but basic characteristics of microarray datasets can be found to identify the potential gene regulatory relationship.</p> <p>Results</p> <p>Based on expression correlation, expression level variation and vectors derived from microarray expression levels, we first introduced several novel parameters to measure the characters of regulating gene pairs. Subsequently, we used the naïve Bayesian network to integrate these features as well as the functional co-annotation between transcription factors and their target genes. Then, based on the character of time-delay from the expression profile, we were able to predict the existence and direction of the regulatory relationship respectively.</p> <p>Conclusions</p> <p>Several novel parameters have been proposed and integrated to identify the regulatory relationship. This new model is proved to be of higher efficacy than that of individual features. It is believed that our parametric approach can serve as a fast approach for regulatory relationship mining.</p

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

Unexpected cell type-dependent effects of autophagy on polyglutamine aggregation revealed by natural genetic variation in C. elegans.

BACKGROUND: Monogenic protein aggregation diseases, in addition to cell selectivity, exhibit clinical variation in the age of onset and progression, driven in part by inter-individual genetic variation. While natural genetic variants may pinpoint plastic networks amenable to intervention, the mechanisms by which they impact individual susceptibility to proteotoxicity are still largely unknown. RESULTS: We have previously shown that natural variation modifies polyglutamine (polyQ) aggregation phenotypes in C. elegans muscle cells. Here, we find that a genomic locus from C. elegans wild isolate DR1350 causes two genetically separable aggregation phenotypes, without changing the basal activity of muscle proteostasis pathways known to affect polyQ aggregation. We find that the increased aggregation phenotype was due to regulatory variants in the gene encoding a conserved autophagy protein ATG-5. The atg-5 gene itself conferred dosage-dependent enhancement of aggregation, with the DR1350-derived allele behaving as hypermorph. Surprisingly, increased aggregation in animals carrying the modifier locus was accompanied by enhanced autophagy activation in response to activating treatment. Because autophagy is expected to clear, not increase, protein aggregates, we activated autophagy in three different polyQ models and found a striking tissue-dependent effect: activation of autophagy decreased polyQ aggregation in neurons and intestine, but increased it in the muscle cells. CONCLUSIONS: Our data show that cryptic natural variants in genes encoding proteostasis components, although not causing detectable phenotypes in wild-type individuals, can have profound effects on aggregation-prone proteins. Clinical applications of autophagy activators for aggregation diseases may need to consider the unexpected divergent effects of autophagy in different cell types

Horizontal transfer, selection and maintenance of antibiotic resistance determinants

Scientific abstract Antibiotic resistance, especially in Gram-negative pathogens, represents a substantial clinical and financial burden to our society. The presented work investigated mechanisms and evolutionary dynamics that promote the emergence and maintenance of resistance in bacteria. In the first study, conserved collateral susceptibility changes were identified across resistant uropathogenic Escherichia coli, which included also changes towards increased sensitivity of these isolates to certain antibiotics. This so-called collateral sensitivity potentiated the effect of antibiotics and prevented the selection of resistant isolates compared to wildtype strains. The mechanism and fitness cost of resistance were important predictors of collateral responses in resistant bacteria, and their rapid clinical identification could inform future evolution-based infection treatment. The second study demonstrated the potential of a transposable element (Tn1) to spread antibiotic resistance during natural transformation of Acinetobacter baylyi. In the course of this horizontal gene transfer mechanism, Tn1-containing DNA entered the bacterial cell, and specific host, as well as transposon proteins, facilitated Tn1-insertion into the recipient chromosome. A mechanistic model of transposition-mediated natural transformation from a circular, cytoplasmic intermediate is presented. In the third study, uropathogenic E. coli improved its permissiveness towards two unrelated multidrug resistance plasmids while adapting to a new environmental niche. Mutations in the CCR and ArcAB regulatory systems resulted in transcriptional downregulation of plasmid genes and explained plasmid cost reduction. The presented evolutionary dynamics improve our understanding of successful associations between bacterial pathogens and resistance plasmids and provide a novel solution to the so-called ‘plasmid paradox’. In a broader perspective, the findings of this thesis advanced our knowledge on the selection, spread and maintenance of antibiotic resistance in bacteria, which is important to counteract its evolution

Natural variation in bacterial gene regulation : a thesis presented in partial fulfilment of the requirements for the degree of Ph.D in Microbiology & Genetics, Massey University College of Science, School of Natural and Computational Sciences

Listed in 2022 Dean's List of Exceptional ThesesFigures 1.1 & 1.2 are re-used with the publishers' permission. Fig 1.5 is re-used under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.It has been over 160 years since Charles Darwin set out the theory of evolution by natural selection. This theory is broadly accepted these days. However, it is still not completely understood how natural selection shapes particular cell mechanisms and behaviours. There is a limited research about selection acting on gene regulation. To address the questions about how selection shapes gene regulation we used a collection of environmental E. coli isolates. We quantified the genetic variability of 605 promoters within this collection of highly diverged strains. We then selected ten promoters (aldA, yhjX, lacZ, aceB, mtr, cdd, dctA, ptsG, purA, and tpiA) which highly differ in their genetic variability to analyse their phenotypic variability. We used fluorescent reporter assays with flow cytometry to measure changes in gene expression with high-throughput and at single cell resolution. In order to discern natural selection acting on gene regulation we compared phenotypes from segregating promoter variants, which have been subject to natural selection and random promoter variants that have never been subject to natural selection. We generated the random variants using PCR random mutagenesis. Beside focusing on the changes in the overall expression (i.e., transcription and translation), we examine selection acting on transcription only. This we achieved be implementing self-cleaving ribozyme insulation. In this thesis we showed that natural selection towards high plasticity and low noise is common among regulated E. coli promoters. We also verify that the self-cleaving ribozyme RiboJ activity is highly effective and that this genetic tool can be used to detect changes in transcription alone. Utilizing the RiboJ we were then able to detect both directional and diversifying selection acting on lacZ promoter. This thesis thus broadens the knowledge about natural selection acting on gene regulation and provides a new insights into how promoters are shaped in nature by selection, including some most well-characterized bacterial promoters. This work also demonstrates a new application of RiboJ ribozyme that has not been published before

Physical, functional and conditional interactions between ArcAB and phage shock proteins upon secretin-induced stress in Escherichia coli

The phage shock protein (Psp) system found in enterobacteria is induced in response to impaired inner membrane integrity (where the Psp response is thought to help maintain the proton motive force of the cell) and is implicated in the virulence of pathogens such as Yersinia and Salmonella. We provided evidence that the two-component ArcAB system was involved in induction of the Psp response in Escherichia coli and now report that role of ArcAB is conditional. ArcAB, predominantly through the action of ArcA regulated genes, but also via a direct ArcB–Psp interaction, is required to propagate the protein IV (pIV)-dependent psp-inducing signal(s) during microaerobiosis, but not during aerobiosis or anaerobiosis. We show that ArcB directly interacts with the PspB, possibly by means of the PspB leucine zipper motif, thereby allowing cross-communication between the two systems. In addition we demonstrate that the pIV-dependent induction of psp expression in anaerobiosis is independent of PspBC, establishing that PspA and PspF can function as a minimal Psp system responsive to inner membrane stress