Biology subjects

Dixon, N.

Publications and source records attributed to Dixon, N..

8 recordsLinked to original sources

bioRxiv2026

Using auxotrophic donor strains to explore pQBR57 plasmid host range among environmental soil bacterial isolates

Plasmid host range (PHR) plays a key role in the spread of ecologically important genes, alongside applications in microbiome engineering, and environmental biotechnology. PHR is a complex trait arising from the combination of plasmid, donor and recipient properties. Most studies of PHR use a single donor strain, leaving the role of the donor unexplored, and often require genetically tagged recipient strains for counter selection, which limits use of non-genetically tractable strains. Here we developed a PHR screening method using auxotrophic donors that bypasses the need to genetically tag recipients, thus allowing the screening of culturable environmental bacterial strains. Specifically, we used two auxotrophic donors (P. fluorescens and P. putida), and the plasmid pQBR57-tphKAB, an environmental plasmid engineered for terephthalic acid bioremediation. We screened a library of 101 soil isolates, as potential recipients, including common soil genera of soil bacteria, Pseudomonas, Bacillus and Xanthomonas. We only observed conjugation into other Pseudomonas, but donor identity affected PHR, with P. fluorescens conjugating the plasmid into more recipient strains than P. putida. Phylogenomic analysis revealed that transconjugants clustered with P. citronellosis and P. putida lineages. In strains that were close relatives of transconjugants but who were unable to acquire the plasmid, we observed 5 defence systems not present in transconjugants that may act as barriers to plasmid acquisition. Our method provides a rapid, tag-free framework for screening PHR in environmental isolates and for investigating the influence of donor identity on plasmid conjugation.

microbiology↗

bioRxiv2024

Genetic bioaugmentation-mediated bioremediation of terephthalate in soil microcosms using an engineered environmental plasmid

Harnessing in situ microbial communities to clean-up polluted natural environments is a potentially efficient means of bioremediation, but often the necessary genes to breakdown pollutants are missing. Genetic bioaugmentation, whereby the required genes are delivered to resident bacteria via horizonal gene transfer, offers a promising solution to this problem. Here we engineered a conjugative plasmid previously isolated from soil, pQBR57, to carry a synthetic set of genes allowing bacteria to consume terephthalate, a chemical component of plastics commonly released during their manufacture and breakdown. Our engineered plasmid caused a low fitness cost and was stably maintained in terephthalate contaminated soil by the bacterium P. putida. Plasmid carriers efficiently bioremediated contaminated soil, achieving complete breakdown of 3.2 mg/g of terephthalate within 8 days. The engineered plasmid horizontally transferred the synthetic operon to P. fluorescens in situ, and the resulting transconjugants degraded 10 mM terephthalate during a 180-hour incubation. Our findings show that environmental plasmids carrying synthetic catabolic operons can be useful tools for in situ engineering of microbial communities to perform clean-up even of complex environments like soil.

synthetic biology↗

bioRxiv2024

Tuning the performance of a TphR-based terephthalate biosensor with a design of experiments approach

Transcription factor-based biosensors are genetic tools that aim to predictability link the presence of a specific input stimuli to a tailored gene expression output. The performance characteristics of a biosensor fundamentally determines its potential applications. However, current methods to engineer and optimise tailored biosensor responses are highly nonintuitive, and struggle to investigate multidimensional sequence/design space efficiently. In this study we employ a design of experiments (DoE) approach to build a framework for efficiently engineering activator-based biosensors with tailored performances, and we apply the framework for the development of biosensors for the polyethylene terephthalate (PET) plastic degradation monomer terephthalate (TPA). We simultaneously engineer the core promoter and operator regions of the responsive promoter, and by employing a dual refactoring approach, we were able to explore an enhanced biosensor design space and assign their causative performance effects. The approach employed here serves as a foundational framework for engineering transcriptional biosensors and enabled development of tailored biosensors with enhanced dynamic range and diverse signal output, sensitivity, and steepness. We further demonstrate its applicability on the development of tailored biosensors for primary screening of PET hydrolases and enzyme condition screening, demonstrating the potential of statistical modelling in optimizing biosensors for tailored industrial and environmental applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/600737v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@2db5d6org.highwire.dtl.DTLVardef@fd0935org.highwire.dtl.DTLVardef@67a01dorg.highwire.dtl.DTLVardef@140da62_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract. Employment of a DoE framework for fine-tuning biosensor performance. C_FIG HighlightsO_LIBioinformatic mining of allosteric transcription factors to produce TPA biosensors C_LIO_LIEfficient sampling of complex sequence-function relationships of genetic circuits C_LIO_LIModelling to learn and optimise biosensor genetic circuits C_LIO_LIApplication of biosensors for primary and secondary enzyme screening applications C_LI

synthetic biology↗

bioRxiv2022

arfA antisense RNA regulates MscL excretory activity

Bacteria adapt to acute changes in their environment by processing multiple input stimuli through signal integration and crosstalk to allow fine tuning of gene expression in response to stress. The response to hypoosmotic shock and ribosome stalling occurs through the action of mechanosensitive channels and ribosome rescue mechanisms respectively. However, it is not known if a mechanistic link exists between these stress response pathways. Here we report that the corresponding Large-conductance mechanosensitive channel (mscL) and Alternative ribosome-rescue factor A (arfA) genes are commonly co-located on the genomes of Gammaproteobacteria and display overlap in their respective 3 UTR and 3 CDS. We show this unusual genomic arrangement permits an antisense RNA mediated regulatory control between mscL and arfA and this modulates MscL excretory activity in E. coli. These findings highlight a mechanistic link between osmotic and translational stress responses in E. coli, and further elucidates the previously unknown regulatory function of arfA sRNA.

molecular biology↗

bioRxiv2021

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD

Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long-lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP-dependent motor protein distantly related to helicases. It was first identified in the model Gram-positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram-positive bacteria. In this work we have undertaken the first comprehensive investigation of the phylogenetic diversity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram-negative bacteria. Therefore, HelD proteins represent an important new class of transcription factor associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

molecular biology↗

bioRxiv2020

Genetic and process engineering strategies for enhanced recombinant N-glycoprotein production in bacteria

BackgroundThe production of N-linked glycoproteins in genetically amenable bacterial hosts offers great potential for reduced cost, faster/simpler bioprocesses, greater customisation and utility for distributed manufacturing of glycoconjugate vaccines and glycoprotein therapeutics. Efforts to optimize production hosts have included heterologous expression of glycosylation enzymes, metabolic engineering, use of alternative secretion pathways, and attenuation of gene expression. However, a major bottleneck to enhance glycosylation efficiency, which limits the utility of the other improvements is the impact of target protein sequon accessibility during glycosylation. ResultsHere, we explore a series genetic and process engineering strategies to increase recombinant N-linked glycosylation mediated by the Campylobacter-derived PglB oligosaccharyltransferase in Escherichia coli. Strategies include increasing membrane residency time of the target protein by modifying the cleavage site of its secretion signal, and modulating protein folding in the periplasm by use of oxygen limitation or strains with compromised oxidoreductases or disulphide-bond isomerase activity. These approaches could achieve up to 90% improvement in glycosylation efficiency. Furthermore, we also demonstrated that supplementation with the chemical oxidant cystine enhanced glycoprotein production and improved cell fitness in the oxidoreductase knock out strain. ConclusionsIn this study, we demonstrated that improved glycosylation in the heterologous host could be achieved by mimicking the coordination between protein translocation, folding and glycosylation observed in native such as Campylobacter jejuni and mammalian hosts. Furthermore, it provides insight into strain engineering and bioprocess strategy, to improve glycoprotein yield and to avoid physiological burden of unfolded protein stress to cell growth. The process and genetic strategies identified herein will inform further optimisation and scale-up of heterologous recombinant N-glycoprotein production

synthetic biology↗

bioRxiv2019

Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

BackgroundGenetically encoded biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcript factors (aTFs). Therefore, altering the substrate specificity of aTFs towards the detection of new effectors is an important goal.\n\nResultsHere, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its substrate specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Substrate recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance.\n\nConclusionsThis is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its substrate specificity to generate a biosensor for aromatic aldehydes.

bioengineering↗

bioRxiv2019

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the bacterial replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of TLS polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

biochemistry↗