Biology subjects

Schniete, J. K.

Publications and source records attributed to Schniete, J. K..

7 recordsLinked to original sources

bioRxiv2023

Standing wave mesoscopy

Standing wave (SW) microscopy is a method that uses an interference pattern to excite fluorescence from labelled cellular structures and produces high-resolution images of three-dimensional objects in a two-dimensional dataset. SW microscopy is performed with high magnification, high numerical aperture objective lenses, and while this results in high resolution images, the field of view is very small. Here we report upscaling of this interference imaging method from the microscale to the mesoscale using the Mesolens, which has the unusual combination of a low magnification and high numerical aperture. With this method, we produce SW images within a field of view of 4.4 mm x 3.0 mm that can readily accommodate over 16,000 cells in a single dataset. We demonstrate the method using both single-wavelength excitation and the multi-wavelength SW method TartanSW. We show application of the method for imaging of fixed and living cells specimens, with the first application of SW imaging to study cells under flow conditions.

cell biology↗

bioRxiv2023

A simple computational method to increase axial contrast in multi-wavelength interference microscopy

Multi-wavelength standing wave (SW) microscopy and interference reflection microscopy (IRM) are powerful techniques that use optical interference to study topographical structure. However, the use of more than two wavelengths to image the complex cell surface results in complicated topographical maps and it can be difficult to resolve the three-dimensional contours. We present a simple image processing method to reduce the thickness and spacing of antinodal fringes in multi-wavelength interference microscopy by up to a factor of two to produce clearer and more precise topographical maps of cellular structures. We first demonstrate this improvement using model non-biological specimens, and we subsequently demonstrate the benefit of our method for reducing the ambiguity of surface topography and revealing obscured features in live and fixed cell specimens.

biophysics↗

bioRxiv2021

Bilateral symmetry of linear streptomycete chromosomes

Here we characterise an uncommon set of telomeres from Streptomyces rimosus ATCC 10970, the parental strain of a lineage of one of the earliest-discovered antibiotic-producers. Following the closure of its genome sequence, we then compared unusual telomeres from this organism with the other five classes of replicon ends found amongst streptomycetes. Closed replicons of streptomycete chromosomes were organised with respect to their phylogeny and physical orientation, which demonstrated that different telomeres were not associated with particular clades and were likely shared amongst different strains by plasmid-driven horizontal gene transfer. Furthermore, we identified a ~50 kb origin island with conserved synteny that is located at the core of all streptomycete chromosomes and forms an axis around which symmetrical chromosome inversions can take place. Despite this chromosomal bilateral symmetry, a bias in parS sites to the right of oriC is maintained across the family Streptomycetaceae and suggests that the formation of ParB/parS nucleoprotein complexes on the right replichore is a conserved feature in streptomycetes. Consequently our studies reveal novel features of linear bacterial replicons that, through their manipulation, may lead to improvements in growth and productivity of this important industrial group of bacteria.

microbiology↗

bioRxiv2020

An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton

Conventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart. Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminated regions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescence microscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs with different axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased the sampling density from 50% to 98% over seven anti-nodal planes, with no notable change in axial or lateral resolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with images of the membrane and cytoskeleton of living and fixed cells.

cell biology↗

bioRxiv2020

ActDES- a Curated Actinobacterial Database for Evolutionary Studies

Actinobacteria are a large and diverse phylum of bacteria that contains medically and ecologically relevant organisms. Many members are valuable sources of bioactive natural products and chemical precursors that are exploited in the clinic. These are made using the enzyme pathways encoded in their complex genomes. Whilst the number of sequenced genomes has increased rapidly in the last twenty years, the large size and complexity of many Actinobacterial genomes means that the sequences remain incomplete and consist of large numbers of contigs with poor annotation, which hinders large scale comparative genomics and evolutionary studies. To enable greater understanding and exploitation of Actinobacterial genomes, specialist genomic databases must be linked to high-quality genome sequences. Here we provide a curated database of 612 high-quality actinobacterial genomes from 80 genera, chosen to represent a broad phylogenetic group with equivalent genome reannotation. Utilising this database will provide researchers with a framework for evolutionary and metabolic studies, to enable a foundation for genome and metabolic engineering, to facilitate discovery of novel bioactive therapeutics and studies on gene family evolution. Significance as a bioresource to the communityThe Actinobacteria are a large diverse phylum of bacteria, often with large, complex genomes with a high G+C content. Sequence databases have great variation in the quality of sequences, equivalence of annotation and phylogenetic representation, which makes it challenging to undertake evolutionary and phylogenetic studies. To address this, we have assembled a curated, taxa-specific, non-redundant database to aid detailed comparative analysis of Actinobacteria. ActDES constitutes a novel resource for the community of Actinobacterial researchers that will be useful primarily for two types of analyses: (i) comparative genomic studies - facilitated by reliable identification of orthologs across a set of defined, phylogenetically-representative genomes, and (ii) phylogenomic studies which will be improved by identification of gene subsets at specified taxonomic level. These analyses can then act as a springboard for the studies of the evolution of virulence genes, the evolution of metabolism and identification of targets for metabolic engineering. Data summaryAll genome sequences used in this study can be found in the NCBI taxonomy browser https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/www.tax.cgi and are summarised along with Accession numbers in Table S1 All other data are available on Figshare https://doi.org/10.6084/m9.figshare.12167529 and https://doi.org/10.5281/zenodo.3830391 O_LIPerl script files available on GitHub https://github.com/nselem/ActDES including details of how to batch annotate genomes in RAST from the terminal https://github.com/nselem/myrast C_LIO_LISupp. Table S1 List of genomes from NCBI (Actinobacteria database.xlsx) https://doi.org/10.6084/m9.figshare.12167529 C_LIO_LICVS genome annotation files including the FASTA files of nucleotide and amino acids sequences (individual .cvs files) https://doi.org/10.6084/m9.figshare.12167880 C_LIO_LIBLAST nucleotide database (.fasta file) https://doi.org/10.6084/m9.figshare.12167724 C_LIO_LIBLAST protein database (.fasta file) https://doi.org/10.6084/m9.figshare.12167724 C_LIO_LISupp. Table S2 Expansion table genus level (Expansion table.xlsx Tab Genus level) https://doi.org/10.6084/m9.figshare.12167529 C_LIO_LISupp. Table S2 Expansion table species level (Expansion table.xlsx Tab species level) https://doi.org/10.6084/m9.figshare.12167529 C_LIO_LIAll GlcP and Glk data - blast hits from ActDES database, MUSCLE Alignment files and .nwk tree files can be found at https://doi.org/10.6084/m9.figshare.12167529 C_LIO_LIInteractive trees in Microreact for Glk tree https://microreact.org/project/w_KDfn1xA/90e6759e and associated files can be found at https://doi.org/10.6084/m9.figshare.12326441.v1 C_LIO_LIInteractive trees in Microreact for GlcP tree https://microreact.org/project/VBUdiQ5_k/0fc4622b and associated files can be found at https://doi.org/10.6084/m9.figshare.12326441.v1 C_LI

microbiology↗

bioRxiv2019

Differential transcription of expanded gene families in central carbon metabolism of Streptomyces coelicolor A3(2)

BackgroundStreptomycete bacteria are prolific producers of specialised metabolites, many of which have clinically relevant bioactivity. A striking feature of their genomes is the expansion of gene families that encode the same enzymatic function. Genes that undergo expansion events, either by horizontal gene transfer or duplication, can have a range of fates: genes can be lost, or they can undergo neo-functionalisation or sub-functionalisation. To test whether expanded gene families in Streptomyces exhibit differential expression, an RNA-Seq approach was used to examine cultures of wild-type Streptomyces coelicolor grown with either glucose or tween as the sole carbon source. ResultsRNA-Seq analysis showed that two-thirds of genes within expanded gene families show transcriptional differences when strains were grown on tween compared to glucose. In addition, expression of specialised metabolite gene clusters (actinorhodin, isorenieratane, coelichelin and a cryptic NRPS) was also influenced by carbon source. ConclusionsExpression of genes encoding the same enzymatic function had transcriptional differences when grown on different carbon sources. This transcriptional divergence enables partitioning to function under different physiological conditions. These approaches can inform metabolic engineering of industrial Streptomyces strains and may help develop cultivation conditions to activate the so-called silent biosynthetic gene clusters.

microbiology↗

bioRxiv2017

Expanding Gene Families Helps Generate The Metabolic Robustness Required For Antibiotic Biosynthesis

Introductory paragraphExpanding the genetic repertoire of an organism by gene duplication or horizontal gene transfer (HGT) can aid adaptation. Streptomyces species are prolific producers of bioactive specialised metabolites with adaptive functions in nature and some have found utility in human medicine such as antibiotics. Whilst the biosynthesis of these specialised metabolites is directed by dedicated biosynthetic gene clusters (BGCs), little attention has been focussed on how these organisms have evolved robustness into their genomes to facilitate the metabolic plasticity required to provide chemical precursors for biosynthesis. Here we show that specific expansions of gene families in central carbon metabolism have evolved and become fixed in Streptomyces bacteria to enable plasticity and robustness that maintain cell functionality whilst costly specialised metabolites are produced. These expanded gene families, in addition to being a metabolic adaptation, make excellent targets for metabolic engineering of industrial specialised metabolite producing bacteria.

microbiology↗