Biology subjects

Lundin, D.

Publications and source records attributed to Lundin, D..

14 recordsLinked to original sources

bioRxiv2026

Mutational and bioinformatic analysis of the binding site for the ribonucleotide reductase-specific transcriptional repressor NrdR

The prevalent transcriptional repressor NrdR binds to highly conserved prokaryotic sequences in the promoter regions of operons encoding the essential enzyme ribonucleotide reductase. The NrdR binding sites consist of two partially palindromic 16 bp sequences (NrdR boxes) separated by a 15-16 bp linker sequence. We have assessed the requirement of both boxes for binding, the propensity of different NrdRs to bind to heterologous binding sites, and that the linker sequence is only limited to length and not sequence conservation. As we have observed several deviations from the conserved sequences of the NrdR boxes, we here test the conservation requirements of individual basepairs in the NrdR boxes using a synthetic DNA fragment (Synt DNA) to which the NrdR proteins from the actinomycete Streptomyces coelicolor and the gammaproteobacterium Escherichia coli bind equally well as to their homologous binding sites. By introducing isolated mutations to Synt DNA and testing the binding capacity of NrdR from S. coelicolor and E. coli we expand our understanding of what criteria are needed to build a functional binding site for the NrdR repressor.

molecular biology↗

bioRxiv2025

nf-core/proteinfamilies: A scalable pipeline for the generation of protein families

The growth of metagenomics-derived amino acid sequence data has transformed our understanding of protein function, microbial diversity and evolutionary relationships. However, the vast majority of these proteins remain functionally uncharacterized. Grouping the millions of such uncharacterised sequences with the few experimentally characterised ones allows the transfer of annotations, while the inspection of conserved residues with multiple sequence alignments can provide clues to function, even in the absence of existing functional information. To address the challenges associated with this data surge and the need to group sequences, we present a scalable, open-source, parametrizable Nextflow pipeline (nf-core/proteinfamilies) that generates protein nascent families or assigns new proteins to existing families. The computational benchmarks demonstrated that resource usage can scale approximately linearly with input size, while the biological benchmarks showed that the generated protein families closely resemble manually curated families found in widely used databases.

bioinformatics↗

bioRxiv2025

The Mycobacterium smegmatis bd-II terminal oxidase employs a 1 carboxylate shift mechanism

Cytochrome bd is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here we characterise the first qOR-2 type bd oxidase, the cyt bd-II from Mycobacterium smegmatis, by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. By over-expressing the appCB operon in its native host, we produce a highly active bd-II (kcat=30 e-s-1) that together with a high-resolution (2.8 [A]) cryo-EM structure and multiscale simulations reveal unique proton pathways and oxygen channels responsible for its function. We propose that O2-scavenging activates a pH-dependent molecular switch, involving coordination changes of heme d and surrounding bulky residues that regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type bd oxidases, and a basis for understanding the evolution of the superfamily.

biochemistry↗

bioRxiv2025

The path to dependence: stepwise genome evolution from a facultative symbiont to an endosymbiont in the N2-fixing diatom-Richelia symbioses

A few genera of diatoms are widespread in the oceans and form stable partnerships with N2-fixing filamentous cyanobacteria Richelia spp. A unique feature of the diatom-Richelia symbioses is the symbiont cellular location spans a continuum of integration (epibiont, periplasmic, endobiont) that is reflected in the symbiont genome size and content. In this study we analyzed genomes derived from cultures and environmental metagenome-assembled genomes focusing on characters indicative of genome evolution. Our results show an enrichment of short length transposable elements (TEs) and pseudogenes in the periplasmic endosymbiont genomes, suggesting an active and transitionary period in genome evolution. In contrast, genomes of endobionts exhibited fewer TEs and pseudogenes, reflecting advanced stages of genome reduction and increased host dependency in the endobionts. Pangenome analyses identified that endobionts streamline their genomes and conserve a majority of their genes in the core, whereas periplasmic endobionts and epibionts maintain larger flexible genomes, suggesting increased genomic plasticity. Functional gene comparisons with other N2-fixing cyanobacteria revealed that Richelia endobionts have similar patterns of metabolic loss but are distinguished by absence of specific pathways (e.g. cytochrome bd ubiquinol oxidase, lipid-A) that increase dependency and direct interaction with their respective hosts. In conclusion, our findings underscore the dynamic nature of genome reduction in N2-fixing cyanobacterial symbionts and demonstrate the diatom-Richelia symbioses as a valuable and rare model to study genome evolution in the transitional stages from a free-living facultative symbiont to a host-dependent endobiont.

microbiology↗

bioRxiv2024

Temperature and dissolved organic matter shape marine prokaryotic activity and gene expression in a sub-arctic sea

Temperature and dissolved organic matter (DOM) are important drivers of microbial activity, but their effects, alone or in combination, on the physiological responses of sub-arctic prokaryotic assemblages remain poorly understood. In a northern Baltic Sea one-month mesocosm experiment, we therefore exposed a coastal microbial community to temperature and nutrient regimes representative of winter and early summer (i.e., 1{degrees}C and 10{degrees}C, with and without DOM additions) in a 2x2 factorial design. Midway through the experiment, specific growth rates were highest for the 10{degrees}C plus DOM treatment (TN; [~]2.5 day-1), comparable for the 1{degrees}C plus DOM (N) and the 10{degrees}C (T) treatments at [~]1.0 day-1; and low for the control (1{degrees}C, no DOM enrichment [C]; 0.2 day-1). Taxonomic analysis of metatranscriptomes uncovered broad treatment specific responses, and a PERMANOVA on the 182,618 transcribed genes revealed statistically significant effects of both temperature and DOM, and significant interaction effects between the two (altogether involving 18% of genes). Significant differences in transcription identified by EdgeR analysis included Nitrosopumilus genes for ammonium uptake and ammonia oxidation in the 1{degrees}C mesocosms (C, N), membrane transporters for small organic acids in the N-treatment, genes for nitrogen and phosphorus assimilation along with molecular chaperones in the T-treatment, and dominance of Oceanospirillales genes for energy and growth metabolism in the TN-treatment. These metatranscriptomic responses were associated with changes in e.g. prokaryotic growth rates and growth efficiency, providing clues to how successional changes in community composition and metabolism are directed by temperature and DOM as central factors underlying environmental change. ImportanceIt is recognized that increases in temperature and dissolved organic matter loading are key to understanding how climate change will influence polar ecosystems. Still, little is known of the effects of these factors on the physiological responses of Arctic prokaryotes. Since prokaryotes are principal drivers of biogeochemical cycles, we investigated how temperature and dissolved organic matter influence prokaryotic transcriptional responses in taxonomy and metabolic pathways. The metatranscriptomics analyses uncovered broad treatment specific responses linked with changes in prokaryotic community composition, growth rates, and growth efficiency. Yet, and importantly, the expression of most metabolic functions remained stable, suggesting a pronounced functional resilience of the prokaryotic community enabled by shifts in the dominance of different taxa. This emphasizes the large potential and importance of identifying the metabolic functions that underlie the divergence of prokaryotic communities in response to environmental changes projected to alter some of the most vulnerable marine environments.

ecology↗

bioRxiv2024

Bacterial transcriptional repressor NrdR - a flexiblemultifactorial nucleotide sensor

NrdR is a bacterial transcriptional repressor consisting of a Zn-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two "NrdR boxes" upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, where we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus dATP or equivalent diphosphate combinations. No other combination of nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR-ATP-dATP and EcoNrdR-ADP-dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-EM structures of DNA-bound EcoNrdR-ATP-dATP and novel filaments of EcoNrdR-ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR-ATP-dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that when loaded with the correct nucleotides adapt to an optimal promoter binding conformation.

biophysics↗

bioRxiv2024

Strong conservation of spacer lengths in NrdR repressor DNA binding sites

The ribonucleotide reductase-specific repressor NrdR, from the human pathogens Listeria monocytogenes and Streptococcus pneumoniae, form tetramers that bind to DNA when loaded with dATP and ATP. If loaded with only ATP they form different oligomeric complexes that cannot bind to DNA. The DNA binding site in L. monocytogenes is a pair of NrdR boxes separated by 15-16 bp, whereas in Streptococcus pneumoniae the NrdR boxes are separated by 25-26 bp. However, Streptococcus pneumoniae NrdR binds stronger to the related Streptococcus thermophilus binding sites with NrdR boxes separated by 15-16 bp. This observation triggered a comprehensive binding study of four NrdRs from L. monocytogenes, Streptococcus pneumoniae, Escherichia coli and Streptomyces coelicolor to a series of synthetic dsDNA fragments where the NrdR boxes were separated by 12-27 bp. All four NrdRs bound well to NrdR boxes separated by 14-17 bp, and also to NrdR boxes separated by 24-27 bp. The worst binding occurred when NrdR boxes were separated by 20 bp. The in vitro results were confirmed in vivo in E. coli for spacer distances 12-27 bp. We conclude that NrdR repressors bind most efficiently when there is an integer number of DNA turns between the center of the two NrdR boxes.

biochemistry↗

bioRxiv2023

Tertiary-interaction characters enable fast, model-based structural phylogenetics beyond the twilight zone

Protein structure is more conserved than protein sequence, and therefore may be useful for phylogenetic inference beyond the "twilight zone" where sequence similarity is highly decayed. Until recently, structural phylogenetics was constrained by the lack of solved structures for most proteins, and the reliance on phylogenetic distance methods which made it difficult to treat inference and uncertainty statistically. AlphaFold has mostly overcome the first problem by making structural predictions readily available. We address the second problem by redeploying a structural alphabet recently developed for Foldseek, a highly-efficient deep homology search program. For each residue in a structure, Foldseek identifies a tertiary interaction closest-neighbor residue in the structure, and classifies it into one of twenty "3Di" states. We test the hypothesis that 3Dis can be used as standard phylogenetic characters using a dataset of 53 structures from the ferritin-like superfamily. We performed 60 IQtree Maximum Likelihood runs to compare structure-free, PDB, and AlphaFold analyses, and default versus custom model sets that include a 3DI-specific rate matrix. Analyses that combine amino acids, 3Di characters, partitioning, and custom models produce the closest match to the structural distances tree of Malik et al. (2020), avoiding the long-branch attraction errors of structure-free analyses. Analyses include standard ultrafast bootstrapping confidence measures, and take minutes instead of weeks to run on desktop computers. These results suggest that structural phylogenetics could soon be routine practice in protein phylogenetics, allowing the re-exploration of many fundamental phylogenetic problems.

evolutionary biology↗

bioRxiv2023

Activity modulation in anaerobic ribonucleotide reductases: nucleotide binding to the ATP-cone mediates long-range order-disorder transitions in the active site

A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding ATP or dATP it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerization and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it can not be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 [A] away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.

biochemistry↗

bioRxiv2021

Taxon-specific shifts in bacterial and archaeal transcription of dissolved organic matter cycling genes in a stratified fjord

A considerable fraction of organic matter derived from photosynthesis in the euphotic zone settles into the oceans interior, and under way is degraded by diverse microbial consortia that utilize a suite of extracellular enzymes and membrane transporters. Still, the molecular details that regulate carbon cycling across depths remain little explored. As stratification in fjords has made them attractive models to explore patterns in biological oceanography, we here analyzed bacterial and archaeal transcription in samples from five depth layers in the Gullmar Fjord, Sweden. Transcriptional variation over depth correlated with gradients in chlorophyll a and nutrient concentrations. Differences in transcription between sampling dates (summer and early autumn), were strongly correlated with ammonium concentrations, which potentially was linked with a stronger influence of (micro-)zooplankton grazing in summer. Transcriptional investment in carbohydrate-active enzymes (CAZymes) decreased with depth and shifted toward peptidases, partly a result of elevated CAZyme transcription by Flavobacteriales, Cellvibrionales and Synechococcales at 2-25 m and a dominance of peptidase transcription by Alteromonadales and Rhodobacterales from 50 m and down. In particular, CAZymes for chitin, laminarin, and glycogen were important. High levels of transcription of ammonium transporters by Thaumarchaeota at depth (up to 18% of total transcription), along with the genes for ammonia oxidation and CO2-fixation, indicated that chemolithoautotrophy contributed to the carbon flux in the fjord. The taxon-specific expression of functional genes for processing of the marine DOM pool and nutrients across depths emphasizes the importance of different microbial foraging mechanisms across spatiotemporal scales for shaping biogeochemical cycles. IMPORTANCEIt is generally recognized that stratification in the ocean strongly influences both the community composition and the distribution of ecological functions of microbial communities, which in turn are expected to shape the biogeochemical cycling of essential elements over depth. Here we used metatranscriptomics analyses to infer molecular detail on the distribution of gene systems central to the utilization of organic matter in a stratified marine system. We thereby uncovered that pronounced shifts in transcription of genes encoding CAZymes, peptidases, and membrane transporters occurred over depth among key prokaryotic orders. This implies that sequential utilization and transformation of organic matter through the water column is a key feature that ultimately influences the efficiency of the biological carbon pump.

ecology↗

bioRxiv2020

Divergent gene expression responses in two Baltic Sea heterotrophic model bacteria to dinoflagellate dissolved organic matter

Phytoplankton release massive amounts of dissolved organic matter (DOM) into the water column during recurring blooms in coastal waters and inland seas. The released DOM encompasses a complex mixture of both known and unknown compounds, and is a rich nutrient source for heterotrophic bacteria. The metabolic activity of bacteria during and after phytoplankton blooms can hence be expected to reflect the characteristics of the released DOM. We therefore investigated if bacterioplankton could be used as "living sensors" of phytoplankton DOM quantity and/or quality, by applying gene expression analyses to identify bacterial metabolisms induced by DOM. We used transcriptional analysis of two Baltic Sea bacterial isolates (Polaribacter sp. BAL334 [Flavobacteriia] and Brevundimonas sp. BAL450 [Alphaproteobacteria]) growing with DOM from axenic cultures of the dinoflagellate Prorocentrum minimum. We observed pronounced differences between the two bacteria both in bacterial growth and the expressed metabolic pathways in cultures exposed to dinoflagellate DOM compared with controls. Differences in metabolic responses between the two isolates were caused both by differences in gene repertoire between them (e.g. in the SEED categories for membrane transport, motility and photoheterotrophy) and the regulation of expression (e.g. fatty acid metabolism), emphasizing the importance of separating the responses of different taxa in analyses of community sequence data. Similarities between the bacteria included substantially increased expression of genes for Ton and Tol transport systems in both isolates, which are commonly associated with uptake of complex organic molecules. Polaribacter sp. BAL334 showed stronger metabolic responses to DOM harvested from exponential than stationary phase dinoflagellates (128 compared to 26 differentially expressed genes), whereas Brevundimonas sp. BAL450 responded more to the DOM from stationary than exponential phase dinoflagellates (33 compared to 6 differentially expressed genes). These findings suggest that shifts in bacterial metabolisms during different phases of phytoplankton blooms can be detected in individual bacterial species and can provide insights into their involvement in DOM transformations.

microbiology↗

bioRxiv2018

Fusion of a functional glutaredoxin to the radical-generating subunit of ribonucleotide reductase

Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyse reduction of the four ribonucleotides to their corresponding deoxyribonucleotides. Facklamia ignava NrdB is an unprecedented fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-cone. Grx, which in general is encoded elsewhere in the genome than is the RNR operon, is a known physiological reductant of RNRs. Here we show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and interestingly also via a monothiol mechanism, although less efficiently. A Grx that utilizes either or of these two reaction mechanisms has to our knowledge not been observed with a native substrate before. The ATP-cone, which is commonly found as an N-terminal domain of the catalytic subunit of RNRs, is an allosteric on/off switch that promotes dNDP reduction in presence of ATP and inhibits the enzyme activity in presence of dATP. Here we show that dATP bound to the ATP-cone of F. ignava NrdB promotes formation of tetramers that are unable to form enzymatically competent complexes with F. ignava NrdA. The ATP-cone binds two molecules of dATP, but only one molecule of the activating nucleotide ATP. F. ignava NrdB contains the recently identified radical factor Mn2III/IV. We show that NrdA from the firmicute F. ignava can form a catalytically competent RNR with the Mn2III/IV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis.

biochemistry↗

bioRxiv2018

Metal-independent ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens

Ribonucleotide reductase (RNR) catalyzes the only known de-novo pathway for production of all four deoxyribonucleotides required for DNA synthesis. In aerobic RNRs, a di-nuclear metal site is viewed as an absolute requirement for generating and stabilizing an essential catalytic radical. Here we describe a new group of RNRs found in Mollicutes, including Mycoplasma pathogens, that possesses a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable DOPA radical species that directly supports ribonucleotide reduction in vitro and in vivo.\n\nThe cofactor synthesis and radical generation processes are fundamentally different from established RNRs and require the flavoprotein NrdI. Several of the pathogens encoding this RNR variant are involved in diseases of the urinary tract and genitalia. Conceivably, this remarkable RNR variant provides an advantage under metal starvation induced by the immune system. We propose that the new RNR subclass is denoted class Ie.

biochemistry↗

bioRxiv2017

Unique ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit

Ribonucleotide reductases (RNRs) are key enzymes in DNA synthesis and repair, with sophisticated allosteric mechanisms controlling both substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leewenhoekiella blandensis radical-generating subunit regulates activity via modifications of quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzyme complexes. The tetramer forms solely by interactions between ATP-cones, as evidenced by a 2.45 [A] crystal structure. We also present evidence for an MnIIIMnIV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by evolutionary capture of the domain by the radical-generating subunit. Our findings present a novel opportunity for dATP-regulation of engineered proteins.

biochemistry↗