Biology subjects

Minton, N. P.

Publications and source records attributed to Minton, N. P..

7 recordsLinked to original sources

bioRxiv2025

Enabling Plasmid-based Expression in Clostridium kluyveri using a Biparental Methylation-Conjugation System

Clostridium kluyveri is a promising biocatalyst for producing medium-chain fatty acids (MCFAs) from waste-derived carbon via chain elongation. MCFAs are platform chemicals with diverse applications across agriculture, food, cosmetics, and fuels, and could support efforts towards tandem resource recovery and sustainable chemical production. However, genetic intractability has hindered efforts to engineer C. kluyveri for improved product yields, control over chain length and selectivity, and production of non-native oleochemicals. Here, we report a streamlined, biparental methylation-conjugation system developed for C. kluyveri DSM555T to bypass the organisms restriction-modification barriers and enable stable plasmid delivery. We use this system to demonstrate heterologous expression of the Fluorescence-Activated absorption-Shifting Tag (FAST), an anaerobic fluorescent reporter. This system supports advances in metabolic engineering of C. kluyveri and the broader adoption of genetic tools in chain elongating bacteria to expand the applications of anaerobic chain elongation in industrial biomanufacturing. Lay SummaryWe developed a streamlined method for genetically modifying Clostridium kluyveri, a promising strain for industrial anaerobic biomanufacturing, and used it to express a fluorescent protein useful for downstream applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/657078v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@11e2a58org.highwire.dtl.DTLVardef@16e1099org.highwire.dtl.DTLVardef@103c72forg.highwire.dtl.DTLVardef@10a1fce_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

A tale of two phage tails: Engineering the host range of bacteriophages infecting Clostridioides difficile

Clostridioides difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea across the globe. Although treatable with a restricted number of antibiotics, the emergence of resistant variants and high relapse rates necessitate alternative countermeasures. Phage therapy represents an attractive option. However, its implementation is handicapped by the narrow host specificity of the C. difficile bacteriophages isolated to date. One strategy to rationally expand phage host range would be to make appropriate modifications to the phage receptor binding protein (RBP). Here, we identify the tail fibre as the RBP of two Myoviridae phages, {Phi}CD1801 and {Phi}CD2301, which were previously isolated and propagated using the C. difficile strains CD1801 (RT078) and CD2301 (RT014), respectively. Contrary to studies into reprogramming the host ranges of phage of other bacterial other species, exchanging the tail fibre genes (tcf/tfp) alone between the two phage was insufficient to change host specificity. Rather, alterations to host range were dependent their exchange together with a putative chaperone encoded by hyp, localised adjacent to the tail fibre gene. Capitalising on this discovery, CRISPR/Cas9 was used to change the host range of one phage to that of the other by swapping the respective tcf/tfp and hyp genes. Significantly, one of the resulting mutants, surpassed both parental phages in terms of host range and efficiency of infection. This is the first time that genome engineering has successfully expanded the host range of a C. difficile phage, a prerequisite for implementing phage for the treatment of CDI. ImportanceAlternatives to antibiotics for treating Clostridioides difficile infection (CDI) are urgently required. Phage therapy presents an attractive option as it has the potential to clear the infection with minimal microbiome disruption and eliminate the possibility of recurrence. However, the C. difficile bacteriophages isolated to date have highly restricted host ranges. Moreover, rational strategies to alter specificity have till now been precluded as the identity of the phage receptor binding proteins involved was largely unknown. Here, we demonstrated that tail fibre proteins and an associated putative chaperone determine the host range of two Myoviridae phage. This enabled the alteration of specificity through CRISPR-mediated genome editing and the creation of a phage derivative with a host range and infection efficiency exceeding that of the parental phages. This is the first time that the host range of a C. difficile phage has been successfully expanded through rational genome engineering.

microbiology↗

bioRxiv2022

Inducible CRISPR/Cas9 allows for multiplexed and rapidly segregated single target genome editing in Synechocystis sp. PCC 6803

Establishing various synthetic biology tools is crucial for the development of cyanobacteria for biotechnology use, especially tools that allow for precise and markerless genome editing in a time-efficient manner. Here we describe a riboswitch-inducible CRISPR/Cas9 system, contained on one single replicative vector, for the model cyanobacteria Synechocystis sp. PCC 6803. A theophylline-responsive riboswitch allowed tight control of Cas9 expression, which enabled reliable transformation of the CRISPR/Cas9 vector into Synechocystis. Induction of the CRISPR/Cas9 mediated various types of genomic edits, specifically deletions and insertions of varying size. The editing efficiency varied depending on the target and intended edit; smaller edits overall performed better, reaching e.g. 100% for insertion of a FLAG-tag onto rbcL. Importantly, the single-vector CRISPR/Cas9 system described herein was also shown to mediate multiplexed editing of up to three targets in parallel in Synechocystis. All single-target and several double-target mutants were also fully segregated after the first round of induction, adding to the usefulness of this system. Further, a vector curing system that is separately induced by nickel and contained on the CRISPR/Cas9 vector itself, improved curing efficiencies by roughly 4-fold, enabling the final mutants to become truly markerless.

synthetic biology↗

bioRxiv2021

Clostridioides difficile binary toxin binding component (CDTb) increases virulence in a hamster model

Clostridioides difficile is the leading cause of hospital-acquired gastrointestinal infection, in part due to the existence of binary toxin (CDT)-expressing hypervirulent strains. We have previously shown that CDT interacts with the TLR2/6 heterodimer to induce inflammation, and in this study we further explore this interaction as well as the contribution of the separate components of CDT, CDTa and CDTb. We found that the binding component, CDTb, is capable of inducing inflammation. Additionally, complementation of a CDT-deficient C. difficile strain with CDTb alone restored virulence in a hamster model of C. difficile infection. Overall, this study demonstrates that the binding component of C. difficile binary toxin contributes to virulence during infection.

microbiology↗

bioRxiv2021

A novel bacteriophage with broad host-range against Clostridioides difficile ribotype 078 elucidates the phage receptor.

Bacteriophage represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent the dysbiosis of the colonic microbiota associated with the treatment of CDI with antibiotics. Whilst numerous phages have been isolated, none have been characterised with broad host-range activity towards PCR ribotype (RT) 078 C. difficile strains despite their considerable relevance to medicine and agriculture. In this study, we isolated four novel C. difficile Myoviruses: {Phi}CD08011, {Phi}CD418, {Phi}CD1801 and {Phi}CD2301. Their characterisation revealed that each was comparable with other C. difficile phages described in the literature, with the exception of {Phi}CD1801 which exhibited a broad host-range activity towards RT 078, infecting 15/16 (93.8%) of the clinical isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. In an attempt to advance these efforts, we conducted a series of fundamental experiments that identified the C. difficile SlpA, the major constituent of the C. difficile surface-layer (S-layer), as the phage receptor. Thus, we demonstrated that {Phi}CD1801 could only bind to RT 012 or RT 027 strains in the presence of a plasmid-borne S-layer cassette corresponding to RT 078. Armed with this information, efforts should now be directed towards the isolation of phages with broad host-range activity against each of the fourteen described S-layer cassette types which could form the basis of an effective cocktail active against a wide range of C. difficile isolates. ImportanceResearch into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridium difficile infection (CDI) is in its infancy, where an optimal "one size fits all" phage cocktail is yet to be derived. The pursuit thus far, has aimed to find phages with the broadest possible host-range. Although, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad-host range activity are yet to be discovered. In this study, we isolate 4 novel Myoviruses including {Phi}CD1801, which exerts the broadest host-range activity towards RT 078 reported in the literature. Through the application of {Phi}CD1801 to robust binding assays, we elucidate SlpA as the phage receptor on the bacterial cell surface. Our finding suggests that an optimal "one size fits all" combinatorial phage cocktail, could theoretically comprise 14 phages, each targeting one of the 14 described S-layer cassettes of C. difficile.

microbiology↗

bioRxiv2021

Application of transposon-insertion sequencing to determine gene essentiality in the acetogen Clostridium autoethanogenum

The majority of the genes present in bacterial genomes remain poorly characterised with up to one third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to easily transformable species leaving those with low DNA-transfer rates out of reach. Here we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as a sole carbon and energy source. IMPORTANCEAlthough microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterised, leaving the function of many as either hypothetical or entirely unknown. High-throughput, transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA-transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here we developed approaches to facilitate transposon-insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases, CO and CO2, into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth providing insights into the utilisation of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon-insertion sequencing to other microbes where DNA-transfer has until now represented a barrier to progress.

molecular biology↗

bioRxiv2017

RNPP-type quorum sensing regulates solvent formation and sporulation in Clostridium acetobutylicum

The strictly anaerobic bacterium Clostridium acetobutylicum is well known for its ability to convert sugars into organic acids and solvents, most notably the potential biofuel butanol. However, the regulation of its fermentation metabolism, in particular the shift from acid to solvent production, remains poorly understood. The aim of this study was to investigate whether cell-cell communication plays a role in controlling the timing of this shift or the extent of solvent formation. Analysis of the available C. acetobutylicum genome sequences revealed the presence of eight putative RNPP-type quorum sensing systems, here designated qssA to qssH, each consisting of RNPP-type regulator gene followed by a small open reading frame encoding a putative signalling peptide precursor. The identified regulator and signal peptide precursor genes were designated qsrA to qsrH and qspA to qspH, respectively. Triplicate regulator mutants were generated in strain ATCC 824 for each of the eight systems and screened for phenotypic changes. The qsrB mutants showed increased solvent formation during early solventogenesis and hence the QssB system was selected for further characterisation. Overexpression of qsrB severely reduced solvent and endospore formation and this effect could be overcome by adding short synthetic peptides to the culture medium representing a specific region of the QspB signalling peptide precursor. In addition, overexpression of qspB increased the production of acetone and butanol and the initial (48-hour) titre of heat-resistant endospores. Together, these findings establish a role for QssB quorum sensing in the regulation of early solventogenesis and sporulation in C. acetobutylicum.

microbiology↗