Biology subjects

Roberts, D. M.

Publications and source records attributed to Roberts, D. M..

12 recordsLinked to original sources

bioRxiv2025

Evaluation of combined root exudate and rhizosphere microbiota sampling approaches to elucidate plant-soil-microbe interaction

O_LIDeciphering the root exudate-driven interplay between plants and the rhizosphere microbiota is essential for understanding plant adaptation to the environment and future-proofing crop production. However, sampling root exudates and rhizosphere soil remains challenging due to the low throughput and destructive nature of the process. C_LIO_LIWe used the staple crop barley [Hordeum vulgare] as a model to benchmark different sampling approaches for simultaneous exudation and microbiota profiling of soil-grown plants. C_LIO_LIExudate profiles and total dissolved organic carbon exudation rates were consistent across different sampling approaches, whereas root biomass, root morphology measurements, and organic nitrogen exudation varied. C_LIO_LIHigh-throughput amplicon sequencing and quantitative PCR (qPCR) of phylogenetic markers and nitrogen cycle-selected genes revealed a protocol-specific footprint in the composition and abundance of rhizosphere bacterial and fungal microbiota. Yet, on average, 75% of microbes enriched in, and differentiating between, barley rhizosphere and unplanted soil controls were recovered across all the sampling approaches evaluated. C_LIO_LIOur results demonstrated that, under the tested conditions, different sampling approaches produced comparable microbiota and exudation patterns, enabling the integrated study of root exudation and microbial profiles from the same plant. The observed differences across sampling approaches must be considered according to the experimental scope. C_LI

plant biology↗

bioRxiv2025

Molecular basis for depsipeptide HDAC inhibitor combinatorial biosynthesis

Polyketides and nonribosomal peptides are important natural product classes with wide-ranging medical and agricultural applications. The analogous enzymatic logic employed by bacterial modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) enables the assembly of hybrid products. One important group of polyketide-nonribosomal peptide hybrids is exemplified by the HDAC-targeting drug romidepsin. This group is assembled by combinatorial biosynthesis involving fusion of a conserved Zn2+-binding pharmacophore to a variable peptide-based cap. Here, we use gene proximity searching to identify the FR-901375 biosynthetic gene cluster in Pseudomonas chlororaphis subsp. piscium DSM 21509. Comparison of the PKS-NRPS encoded by this gene cluster with those assembling related depsipeptide HDAC inhibitors suggests a novel subunit docking modality enables interaction between the conserved pharmacophore and variable cap biosynthetic machineries. This hypothesis was validated using crosstalk assays, mutagenesis, AlphaFold predictions, and carbene footprinting, providing new insight into the evolution of mechanisms for hybrid polyketide-nonribosomal peptide combinatorial biosynthesis.

biochemistry↗

bioRxiv2025

Nitrogen deletion in the biosynthesis of HDAC-targeting anticancer drugs

Small molecules play indispensable roles in living systems as hormones, membrane bilayer constituents, enzyme cofactors, metal chelators, and defensive chemicals. The exceptional structural diversity of such molecules underpins their wide-ranging biological functions. Precise functional group insertion into various molecular scaffold classes is a hallmark of small molecule biosynthesis and is frequently important for biological activity. Functional group deletion is also important, but mechanisms are less well understood. Here, we report deletion of a cysteine-derived nitrogen atom during assembly of the conserved pharmacophore in the anticancer drug romidepsin, and related depsipeptide HDAC inhibitors. A shunt metabolite hydroxylated at the cysteine--carbon-derived position is a thousand-fold less active, indicating nitrogen deletion is important for potent HDAC inhibition. In vitro reconstitution and dissection of the complete nonribosomal peptide synthetase-polyketide synthase-mediated pathway for pharmacophore assembly reveal that cryptic S-octanoylation is catalysed by an atypical heterocyclisation domain, while multifunctional dehydratase and ketoreductase domains and trans-acting phosphotransferase and flavin-dependent oxidoreductase enzymes catalyse successive transformations in nitrogen deletion. Our findings significantly advance the understanding of heteroatom deletion mechanisms in small molecule biosynthesis and highlight the key role this can play in enhancing bioactivity. One-pot biocatalytic synthesis of the pharmacophore provides foundations for chemoenzymatic approaches to next-generation HDAC inhibitors.

biochemistry↗

bioRxiv2023

Complement-mediated killing of bacteria by mechanical destabilization of the cell envelope

Complement proteins eliminate Gram-negative bacteria in serum via the formation of membrane attack complex (MAC) pores in the outer membrane. However, it remains unclear how outer membrane poration leads to inner membrane permeation and cell lysis. Using atomic force microscopy (AFM) on living Escherichia coli (E. coli), we probed MAC-induced changes in the cell envelope and correlated these with subsequent cell death. Initially, bacteria survived despite the formation of hundreds of MACs randomly distributed over the cell surface. This was followed by larger-scale disruption of the outer membrane, including propagating defects and fractures, and by an overall swelling and stiffening of the bacterial surface, which precede inner membrane permeation. We conclude that bacterial cell lysis is only an indirect effect of MAC formation; outer membrane poration leads to mechanical destabilization of the cell envelope, reducing its ability to contain the turgor pressure, causing inner membrane permeation and cell death. This represents a previously unknown route to bacterial cell death that could be exploited by novel antibiotic treatments.

microbiology↗

bioRxiv2023

Reliable detection and quantification of plasmodesmal callose in Nicotiana benthamiana leaves during defense responses

Callose, a beta-(1,3)-D-glucan polymer, is essential for regulating intercellular trafficking via plasmodesmata (PD). Pathogens manipulate PD-localized proteins to enable intercellular trafficking by removing callose at PD, or conversely by increasing callose accumulation at PD to limit intercellular trafficking during infection. Plant defense hormones like salicylic acid regulate PD-localized proteins to control PD and intercellular trafficking during innate immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing the intercellular trafficking activity during plant immunity. Despite the popularity of this metric there is no standard for how these measurements should be made. In this study, three commonly used methods for identifying and quantifying PD callose by aniline blue staining were evaluated to determine the most effective in the Nicotiana benthamiana leaf model. The results reveal that the most reliable method used aniline blue staining and fluorescent microscopy to measure callose deposition in fixed tissue. Manual or semi-automated workflows for image analysis were also compared and found to produce similar results although the semi-automated workflow produced a wider distribution of data points.

plant biology↗

bioRxiv2023

Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Our data, thus, demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

microbiology↗

bioRxiv2022

Differential Boric Acid and Water Transport in Type I and Type II Pores of Arabidopsis Nodulin 26-Intrinsic Protein

Nodulin-26 intrinsic proteins (NIPs) are plant-specific multifunctional aquaporin-like channels that are phylogenetically and structurally segregated into three subfamilies: NIP I, II, and III. Each subfamily has a characteristic selectivity filter sequence (the "aromatic-arginine" region, or ar/R) that controls substrate transport specificity based on steric constraints, hydrophobicity, and the spatial orientation of hydrogen bonding moieties. All three NIP subfamilies transport metalloid hydroxides, both beneficial as well as toxic, but with different selectivities. Here we investigated the B, As, and water selectivity of representative Arabidopsis thaliana NIP I and II proteins as well as their ar/R mutants in transport assays as well as through B complementation analysis in the B sensitive nip5;1 mutant background. All NIP proteins, and their ar/R mutants, showed equal permeability to arsenite, but showed differences in boric acid and aquaporin activities that was linked to the amino acid at the helix 2 (H2) position of the ar/R filter (Ala for NIP II and Trp for NIP I). The presence of an alanine at this position in NIP II proteins enhances boric acid permeability and drastically reduces the aquaporin/water permeability of the channel. A NIP II structural model generated from the AlphaFold2 resource and evaluated by MD simulation shows that the alanine results in a wider ar/R pore that accommodates the trigonal boric acid molecule and may allow gating of the pore in a manner that affects water permeability. In contrast, NIP I proteins adopt a more classical aquaporin/glyceroporin arrangement in the ar/R that allows metalloid permeability, although with greater selectivity, as well as permeation by water.

biochemistry↗

bioRxiv2021

Chromosome remodelling by SMC/Condensin in B. subtilis is regulated by Soj/ParA during growth and sporulation

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving ATP-dependent dimerization and DNA binding, leading to chromosome segregation and SMC loading. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation, and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. We now show that a major redistribution of SMC complexes drives axial filament formation, in a process regulated by ParA/Soj. Unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyse ATP. These results reveal a new role for ParA/Soj proteins in the regulation of SMC dynamics in bacteria, and yet further complexity in the web of interactions involving chromosome replication, segregation, and organization, controlled by ParAB and SMC.

microbiology↗

bioRxiv2021

The Arabidopsis Calcium Sensor Calmodulin-like 38 Regulates Stress Granule Autophagy and Dynamics during Low Oxygen Stress and Re-aeration Recovery

In response to the energy crisis resulting from submergence stress and hypoxia, Arabidopsis limits non-essential mRNAs translation, and accumulate cytosolic stress granules (SG). SGs are phase-separated mRNA-protein particles that partition transcripts for various fates: storage, degradation, or return to translation after stress alleviation. Here, it is shown that RNA stress granules are dynamically regulated during hypoxia stress and aerobic recovery via two phases of autophagy that require the AAA+ ATPase CDC48 and the calcium sensor Calmodulin-like 38 (CML38). CML38 is a core hypoxia response-protein that associates with hypoxia-induced SGs. We show that CML38 is essential for SG autophagy during extended hypoxia. Further, cml38 mutants show disorganized SG morphology during extended hypoxia, suggesting a role in SG formation and maintenance. We also show that upon the return of aerobic conditions, intracellular calcium and CML38 are necessary for SG breakdown and turnover, and for upregulating autophagy. cml38 mutants not only lose these responses, but also have aberrant, sustained autophagosome accumulation during the reoxygenation recovery phase. The findings suggest that CDC48 RNA granule autophagy ("granulophagy") is conserved in plants, and that the hypoxia-induced calcium sensor CML38 regulates SG autophagy during anaerobic stress as well as during the reprogramming phase associated with reoxygenation.

plant biology↗

bioRxiv2020

The Arabidopsis thaliana NIP2;1 Lactic Acid Channel promotes Plant Survival Under Low Oxygen Stress

Under anaerobic stress Arabidopsis thaliana induces the expression of a collection of core hypoxia genes that encode proteins associated with an adaptive response. Included in these core hypoxia genes is NIP2;1, which encodes a member of the "Nodulin-like Intrinsic Protein" (NIP) subgroup of the aquaporin superfamily of membrane channel proteins. Under normal growth, NIP2;1 expression is limited to the "anoxia core" region of the root stele, but shows substantial induction in response to low oxygen stress (as high as 1000-fold by 2-4 hr of hypoxia challenge), and accumulates in all root tissues. During hypoxia, NIP2;1-GFP, accumulates on the cell surface by 2 hr and then is distributed between the cell surface and internal membranes during sustained hypoxia, and remains elevated in root tissues through 4 hrs of reoxygenation recovery. T-DNA insertional mutant nip2;1 plants show elevation of lactic acid within root tissues, and a reduced efflux of lactic acid and acidification of the external medium. Together with previous biochemical evidence demonstrating that NIP2;1 has lactic acid permease activity, the present work supports the hypothesis that the protein facilitates the release of cellular lactate to the rhizosphere to prevent lactic acid toxicity. In support of this, nip2;1 plants show poorer survival to argon-induced hypoxia stress. Nip2;1 mutant plants also show elevated expression of ethanolic fermentation transcripts, as well as reduced expression the lactate metabolic enzyme GOX3, suggesting that the altered efflux of lactate through NIP2;1 regulates other pyruvate and lactate metabolism pathways. One-sentence SummaryThe NIP2;1 lactic acid permease is necessary for an optimum response to low oxygen stress through the release of lactate from roots during hypoxia stress.

plant biology↗

bioRxiv2019

Regulator of Gene Silencing-Calmodulin associates with mRNA granules and the autophagy protein ATG8

Abstract Regulator-of-gene-silencing calmodulins (rgsCaM) represent a phylogenetic subfamily of calmodulin-like calcium sensors that are targets of viral induced suppression of posttranscriptional gene silencing by secondary siRNAs. The present work shows that a stress (hypoxia) that induces mRNP granule formation also induces the relocalization of rgsCaM to cytosolic granule-like foci that interact with the surface of stress granule and processing body structures. Co-expression of rgsCaM and its binding protein Suppressor of Gene Silencing 3 causes re-localization and integration of rgsCaM into stress granule structures. RgsCaMs contain a conserved topology that consists for four EF hand like domains (three functional and one divergent) that are separated into two calcium binding lobes with an extended amino terminal region. RgsCaM also contains an "ATG8 family interacting motif" (AIM) within its amino-terminal domain that is characteristic of selective autophagy cargo receptors. Co-localization experiments and ratiometric BiFC analyses in Nicotiana benthamiana support the hypothesis that rgsCaM binds directly to ATG8e through this conserved AIM domain, and the two proteins co-localize with mRNP granule markers. Previous reports show that rgsCaM mediates the suppression of gene silencing, at least in part, via turnover of SGS3 via autophagy. A model is proposed for rgsCaM-like proteins as potential mediators of selective autophagy of RNA granules in response to biotic and abiotic stresses.

plant biology↗

bioRxiv2018

A high-throughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis

Tuberculosis is a disease of global importance for which novel drugs are urgently required. We developed a whole-cell phenotypic screen which can be used to identify inhibitors of Mycobacterium tuberculosis growth. We used recombinant strains of virulent M. tuberculosis which express far-red fluorescent reporters and used fluorescence to monitor growth in vitro. We optimized our high throughput assays using both 96-well and 384-well plates; both formats gave assays which met stringent reproducibility and robustness tests. We screened a compound set of 1105 chemically diverse compounds previously shown to be active against M. tuberculosis and identified primary hits which showed [≥] 90% growth inhibition. We ranked hits and identified three chemical classes of interest - the phenoxyalkylbenzamidazoles, the benzothiophene 1-1 dioxides, and the piperidinamines. These new compound classes may serve as starting points for the development of new series of inhibitors that prevent the growth of M. tuberculosis. This assay can be used for further screening, or could easily be adapted to other strains of M. tuberculosis.

microbiology↗