Biology subjects

Thomas, C. M.

Publications and source records attributed to Thomas, C. M..

7 recordsLinked to original sources

bioRxiv2025

LGBTQ+ realities in the biological sciences

While scientific environments have been described as unwelcoming to the LGBTQ+ community, and fields such as physics have systematically documented these challenges, the climate in biology workplaces has not been assessed. We conducted the largest survey to date of LGBTQ+ biologists to examine how their sense of belonging and perception of climate in the biology workplace and professional societies compare to that of their straight and cis peers. We surveyed 1419 biologists across five professional societies, with 486 identifying as LGBTQ+. Trans and gender non-conforming (TGNC) biologists reported lower belonging and morale within the workplace, professional societies, and the biology community compared to cis, straight biologists. They also reported being less comfortable with the climate of various professional biology environments. While LGBTQ+ biologists report that their workplaces are moderately inclusive, over 20% of all LGBTQ+ biologists and nearly 40% of TGNC biologists experience exclusionary behavior at work. This landmark survey provides the first comprehensive analysis of the LGBTQ+ climate in biology, revealing specific challenges faced by TGNC scientists and identifying interventions to enhance inclusivity for scientists. Significance StatementThis landmark study includes the largest known sample of LGBTQ+ biologists and offers the first comprehensive description of the LGBTQ+ climate in biology, differentiating between the experiences of cisgender lesbian, gay, bisexual, and queer (LGBQ) biologists and transgender and gender non-conforming (TGNC) biologists. The study found that compared to non-LGBTQ+ biologists, TGNC participants report lower belonging, morale and comfort with the climate across biology workplaces, professional societies, and the biology community. While on average LGBTQ+ participants reported that their workplaces are moderately inclusive, over 20% of all LGBTQ+ biologists and nearly 40% of TGNC biologists report experiencing exclusionary behaviors at work. The study offers immediate implications for institutional policies and professional development in the biological sciences.

scientific communication and education↗

bioRxiv2024

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Long-range gene regulation is rare in bacteria and is confined to the classical DNA looping model. Here, we use a combination of biophysical approaches, including X-ray crystallography and single-molecule analysis, to show that long-range gene silencing on the plasmid RK2, a source of multidrug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We find that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed-clamp state. KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Altogether, our findings explain the mechanistic basis for KorB role-switching from a DNA-sliding clamp to a co-repressor, and provide a new paradigm for the long-range regulation of gene expression.

microbiology↗

bioRxiv2024

Evolution of a Plasmid Regulatory Circuit Ameliorates Plasmid Fitness Cost

Plasmids play a major role in rapid adaptation of bacteria by facilitating horizontal transfer of diverse genes, most notably those conferring antibiotic resistance. While most plasmids that replicate in a broad range of bacteria also persist well in diverse hosts, there are exceptions that are poorly understood. We investigated why a broad-host range plasmid, pBP136, originally found in clinical Bordetella pertussis isolates, quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution we found that inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid maintenance in E. coli. This gene inactivation resulted in decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. It also caused transcriptional changes in many chromosomal genes primarily related to metabolism. In silico analyses suggested that the change in plasmid transcriptome may be initiated by Upf31 interacting with the plasmid regulator KorB. Expression of upf31 in trans negatively affected persistence of pBP136{Delta}upf31 as well as the closely related archetypal IncP-1{beta} plasmid R751, which is stable in E. coli and natively encodes a truncated upf31 allele. Our results demonstrate that while the upf31 allele in pBP136 might advantageously modulate gene expression in its original host, B. pertussis, it has harmful effects in E. coli. Thus, evolution of a single plasmid gene can change the range of hosts in which that plasmid persists, due to effects on the regulation of plasmid gene transcription.

microbiology↗

bioRxiv2021

Extending the application of the SCA/Sectors method for the identification of domain boundaries and subtype specific residues in multi-domain biosynthetic proteins: Application to Polyketide Synthases

Polypeptides with multiple enzyme domains, such as type I polyketide synthases, produce chemically complex compounds that are difficult to produce via conventional chemical synthesis and are often pharmaceutically or otherwise commercially valuable. Engineering polyketide synthases, via domain swapping and/or site directed mutagenesis, in order to generate novel polyketides, has tended to produce either low yields of product or no product at all. The success of such experiments may be limited by our inability to predict the key functional residues and boundaries of protein domains. Computational tools to identify the boundaries and the residues determining the substrate specificity of domains could reduce the trial and error involved in engineering multi-domain proteins. In this study we use statistical coupling analysis to identify networks of co-evolving residues in type I polyketide synthases, thereby predicting domain boundaries. We extend the method to predicting key residues for enzyme substrate specificity. We introduce bootstrapping calculations to test the relationship between sequence length and the number of sequences needed for a robust analysis. Our results show no simple predictor of the number of sequences needed for an analysis, which can be as few as a hundred and as many as a few thousand. We find that polyketide synthases contain multiple networks of co-substituting residues: some are intradomain but most multiple domains. Some networks of coupled residues correlate with specific functions such as the substrate specificity of the acyl transferase domain, the stereo chemistry of the ketoreductase domain, or domain boundaries that are consistent with experimental data. Our extension of the method provides a ranking of the likely importance of these residues to enzyme substrate specificity, allowing us to propose residues for further mutagenesis work. We conclude that analysis of co-evolving networks of residues is likely to be an important tool for re-engineering multi-domain proteins. Author summaryMany important compounds such as antibiotics or food flavourings are produced naturally by molecular factories within plant, fungal and bacterial cells. These molecular factories typically comprise a complex of multiple interacting enzymes, each enzyme being a stage in a molecular production line. Often the enzymes are connected together as subsections of the same amino acid chain, i.e. protein, with the amino acid chain folding into the separate functional enzymatic domains that comprise the production line. Polyketide synthases are such multi-domain proteins, and their products often have antibacterial, antifungal and antitumoric effects. Engineering polyketide synthases thus has the potential to produce novel drug candidates. We applied and developed statistical approaches to detect where in an amino acid sequence the boundaries are between different domains, potentially allowing these regions to be swapped around for the synthesis of novel compounds. We used the same approaches to identify parts of the amino acid chain important for the function of different types of domain, pointing to how they might be modified to make novel compounds. These analyses agree with published experimental data and allow us to make novel predictions, which we expect to help experimentalists produce novel compounds of commercial and pharmaceutical interest.

bioengineering↗

bioRxiv2020

The Drosophila EGFR ligand mSpitz is delivered to cytoplasmic capes at sites of non-canonical RNA export on the nuclear envelope via the endosomal system

Nuclear-cytoplasmic communication is not limited to nuclear pores, with both proteins and RNA using alternative routes between these compartments. We previously characterized cytoplasmic capes (large invaginations of the nuclear envelope in Drosophila), which are enriched for the membrane-bound EGF receptor ligand mSpitz, endosome-related organelles and ubiquitylated proteins. Closely associated with capes are groups of perinuclear vesicles resembling those seen at sites of RNP export via a budding mechanism. Here, we demonstrate that mSpitz delivery to capes requires passage through the endosomal system. We also show that capes are closely associated with sites of non-canonical RNP export as well as the dFrizzled2 receptor C terminal fragment, a core component of this export pathway. Video microscopy of glands in intact larvae indicates that cytoplasmic capes are stable structures that persist for at least 90 minutes without conspicuous growth. We further show that capes appear with the growth of the salivary gland rather than its developmental stage. Finally, we show that the large F-actin binding protein {beta}H-spectrin, which modulates endosomal trafficking, as well as its partner -spectrin are required for cape formation. Cytoplasmic capes therefore represent a subspecialization of the nuclear envelope where endosomal trafficking and RNP export are closely associated. SynopsisWe further characterize large invaginations of the nuclear envelope called cytoplasmic capes in Drosophila. The EGF receptor ligand mSpitz is concentrated in capes and we show that it traffics to this compartment via endosomes. The presence of RNP and the dFrizzled2 receptor C-terminal fragment also indicates that non-canonical RNA export is concentrated at capes. In vivo imaging shows that capes persist for at least 90 minutes. Finally, the large F-actin crosslinker /{beta}{square}-spectrin is shown to be required for cape formation. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/203000v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@55435borg.highwire.dtl.DTLVardef@1b92320org.highwire.dtl.DTLVardef@1b13af8org.highwire.dtl.DTLVardef@96ab1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

Genes encoding recognition of the Cladosporium fulvum effector protein Ecp5 are encoded at several loci in the tomato genome

The molecular interactions between tomato and Cladosporium fulvum have been an important model for molecular plant pathology. Complex genetic loci on tomato chromosomes 1 and 6 harbor genes for resistance to Cladosporium fulvum, encoding receptor like-proteins that perceive distinct Cladosporium fulvum effectors and trigger plant defenses. Here, we report classical mapping strategies for loci in tomato accessions that respond to Cladosporium fulvum effector Ecp5, which is very sequence-monomorphic. We screened 139 wild tomato accessions for an Ecp5-induced hypersensitive response, and in five accessions, the Ecp5-induced hypersensitive response segregated as a monogenic trait, mapping to distinct loci in the tomato genome. We identified at least three loci on chromosomes 1, 7 and 12 that harbor distinct Cf-Ecp5 genes in four different accessions. Our mapping showed that the Cf-Ecp5 in Solanum pimpinellifolium G1.1161 is located at the Milky Way locus. The Cf-Ecp5 in Solanum pimpinellifolium LA0722 was mapped to the bottom arm of chromosome 7, while the Cf-Ecp5 genes in Solanum lycopersicum Ontario 7522 and Solanum pimpinellifolium LA2852 were mapped to the same locus on the top arm of chromosome 12. Bi-parental crosses between accessions carrying distinct Cf-Ecp5 genes revealed putative genetically unlinked suppressors of the Ecp5-induced hypersensitive response. Our mapping also showed that Cf-11 is located on chromosome 11, close to the Cf-3 locus. The Ecp5-induced hypersensitive response is widely distributed within tomato species and is variable in strength. This novel example of convergent evolution could be used for choosing different functional Cf-Ecp5 genes according to individual plant breeding needs.

genetics↗

bioRxiv2018

DeepCDpred: Inter-residue Distance and Contact Predictionfor Improved Prediction of Protein Structure.

Rapid, accurate prediction of protein structure from amino acid sequence would accelerate fields as diverse as drug discovery, synthetic biology and disease diagnosis. Massively improved prediction of protein structures has been driven by improving the prediction of the amino acid residues that contact in their 3D structure. For an average globular protein, around 92% of all residue pairs are non-contacting, therefore accurate prediction of only a small percentage of inter-amino acid distances could increase the number of constraints to guide structure determination. We have trained deep neural networks to predict inter-residue contacts and distances. Distances are predicted with an accuracy better than most contact prediction techniques. Addition of distance constraints improved de novo structure predictions for test sets of 158 protein structures, as compared to using the best contact prediction methods alone. Importantly, usage of distance predictions allows the selection of better models from the structure pool without a need for an external model assessment tool. The results also indicate how the accuracy of distance prediction methods might be improved further.

bioinformatics↗