Biology subjects

Herrmann, J.

Publications and source records attributed to Herrmann, J..

29 records · Page 2Linked to original sources

bioRxiv2022

Deep haplotype analyses of target-site resistance locus ACCase in blackgrass enabled by pool-based amplicon sequencing

Rapid adaptation of weeds to herbicide applications in agriculture through resistance development is a widespread phenomenon. In particular, the grass Alopecurus myosuroides is an extremely problematic weed in cereal crops with the potential to manifest resistance in the course of only a few generations. Target-site resistances (TSRs), with their strong phenotypic response, play an important role in this rapid adaptive response. Recently, using PacBios long-read amplicon sequencing technology in hundreds of individuals, we were able to decipher the genomic context in which TSR mutations occur. However, sequencing individual amplicons is both costly and time consuming, thus impractical to implement for other resistance loci or applications. Alternatively, pool-based approaches overcome these limitations and provide reliable allele frequencies, albeit at the expense of not preserving haplotype information. In this proof-of-concept study, we sequenced with PacBio High Fidelity (HiFi) reads long-range amplicons (13.2 kb) encompassing the entire ACCase gene in pools of over hundred individuals, and resolved them into haplotypes using the clustering algorithm PacBio amplicon analysis (pbaa), a new application for pools and for plants. From these amplicon pools, we were able to recover most haplotypes from previously sequenced individuals of the same population. In addition, we analyzed new pools from a Germany-wide collection of A. myosuroides populations and found that TSR mutations originating from soft sweeps of independent origin were common. Forward-in-time simulations indicate that TSR haplotypes will persist for decades even at relatively low frequencies and without selection, pointing to the importance of accurate measurement of TSR haplotype prevalence for weed management.

genomics↗

bioRxiv2022

Interference with lipoprotein maturation sensitizes methicillin-resistant Staphylococcus aureus to human group IIA secreted phospholipase A2 and daptomycin.

Methicillin-resistant Staphylococcus aureus (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA secreted phospholipase A2 (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including S. aureus. To determine hGIIA-resistance mechanisms of MRSA we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of lspA, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the lspA mutant was associated with faster and increased cell wall penetration of hGIIA. Moreover, lspA deletion also increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. Exposure of MRSA wild-type to the LspA-specific inhibitors globomycin and myxovirescin A1 induced a lspA mutant phenotype with regard to hGIIA and daptomycin killing. Analysis of >26,000 S. aureus genomes showed that LspA is highly sequence-conserved, suggesting that LspA inhibition could be applied universally. The role of LspA in hGIIA resistance was not restricted to MRSA since Streptococcus mutans and Enterococcus faecalis were also more hGIIA-susceptible after lspA deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological blocking of LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically-applied antibiotics.

microbiology↗

bioRxiv2022

Global analysis of biosynthetic gene clusters reveals conserved and unique natural products in entomopathogenic nematode-symbiotic bacteria

Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus (XP) living in mutualistic symbiosis with entomopathogenic nematodes produce a myriad of natural products to mediate bacteria-nematode-insect interactions. However, a lack of systematic analysis of the biosynthetic gene clusters (BGCs) has limited the understanding of how natural products justify the bacterial niche specificity. Here we combine pangenome and sequence similarity networks to analyze BGCs from 45 XP species. The identified 1,000 BGCs belong to 176 families, over half of which are unknown. Eleven BGCs represent the most conserved families. We then homologously express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, insect virulence factors, or insect immune suppressors. These findings account for the functional basis of bacterial natural products in this tripartite relationship.

biochemistry↗

bioRxiv2021

The mycobacterial mutasome: composition and recruitment in live cells

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded "mycobacterial mutasome" - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III {beta} subunit ({beta} clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted {beta} clamp-binding motif abolishes ImuB-{beta} clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this {beta} clamp-binding antibiotic collapses pre-formed ImuB-{beta} clamp complexes. These observations establish the essentiality of the ImuB-{beta} clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

microbiology↗

bioRxiv2021

Gaining or cutting SLAC: the evolution of plant guard cell signalling pathways

The evolution of adjustable plant pores (stomata), enabling CO2 acquisition in cuticle wax-sealed tissues was one of the most significant events in the development of life on land. But how did the guard cell signalling pathways that regulate stomatal movements evolve? We investigate this through comparison of fern and angiosperm guard cell transcriptomes. We find that these divergent plant groups share expression of similar genes in guard cells including biosynthesis and signalling genes for the drought stress hormone abscisic acid (ABA). However, despite conserved expression in guard cells, S-type anion channels from the SLAC/SLAH family - known for ABA-mediated stomatal closure in angiosperms - are not activated by the same pathways in ferns, highlighting likely differences in functionality. Examination of other land plant channels revealed a complex evolutionary history, featuring multiple gains or losses of SLAC activation mechanisms, as these channels were recruited to a role in stomatal closure. Taken together, the guard cells of flowering and non-flowering plants share similar core features, but also show lineage-specific and ecological niche-related adaptations, likely underlying differences in behaviour.

plant biology↗

bioRxiv2020

Analysis of translating mitoribosome reveals functional characteristics of protein synthesis in mitochondria of fungi

Mitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight in to the biogenesis of the mitoribosomal large subunit and translation regulation.

biochemistry↗

bioRxiv2019

GABA signalling in guard cells acts as a 'stress memory' to optimise plant water loss

The non-protein amino acid {gamma}-aminobutyric acid (GABA) has been proposed to be an ancient messenger for cellular communication conserved across biological kingdoms. GABA has well-defined signalling roles in animals; however, whilst GABA accumulates in plants under stress it has not been determined if, how, where and when GABA acts as an endogenous plant signalling molecule. Here, we establish that endogenous GABA is a bona fide plant signal, acting via a mechanism not found in animals. GABA antagonises stomatal movement in response to opening and closing stimuli in multiple plant families including dicot and monocot crops. Using Arabidopsis thaliana, we show guard cell GABA production is necessary and sufficient to influence stomatal aperture, transpirational water loss and drought tolerance via inhibition of stomatal guard cell plasma membrane and tonoplast-localised anion transporters. This study proposes a novel role for GABA - as a stress memory - opening new avenues for improving plant stress tolerance.

plant biology↗

bioRxiv2019

Deconstructing the structural conservation of distantly related bacterial nucleoid-associated proteins using functional chimeras

Nucleoid-associated proteins (NAPs) are DNA-binding proteins critical for the organization and function of the bacterial chromosome. A subclass of NAPs, including Caulobacter crescentus GapR and Escherichia coli H-NS, preferentially bind AT-rich regions of the nucleoid, but phylogenetic groups that encode GapR rarely encode H-NS. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recent DNA-bound crystal structure of GapR (Guo et al, 2018), we show that although evolutionarily distant, GapR and H-NS possess two regions that are structurally and functionally conserved. These regions are involved in self-association and DNA-binding, even though the two proteins oligomerize and regulate transcription differently. Functional analysis of GapR and H-NS protein chimeras identified structural elements present in H-NS but absent in GapR that rationalize differences in transcriptional regulation. In addition, we identified a sequence element unique to GapR that enables assembly into its tetrameric state. Using fluid-atomic force microscopy, we showed that GapR is capable of bridging DNA molecules in vitro. Together, these results demonstrate that two distantly related NAPs utilize evolutionarily conserved structural elements to serve specialized cellular roles via distinct mechanisms.

molecular biology↗

bioRxiv2019

A Bacterial Surface Layer Protein Exploits Multi-step Crystallization for Rapid Self-assembly

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the C. crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multi-step assembly pathway. This pathway involves two domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2D crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using time-resolved electron cryo-microscopy (Cryo-EM) reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the two domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.\n\nSignificance StatementMany microbes assemble a crystalline protein layer on their outer surface as an additional barrier and communication platform between the cell and its environment. Surface layer proteins efficiently crystallize to continuously coat the cell and this trait has been utilized to design functional macromolecular nanomaterials. Here, we report that rapid crystallization of a bacterial surface layer protein occurs through a multi-step pathway involving a crystalline intermediate. Upon calcium-binding, sequential changes occur in the structure and arrangement of the protein, which are captured by time-resolved small angle x-ray scattering and transmission electron cryo-microscopy. We demonstrate that a specific domain is responsible for enhancing the rate of self-assembly, unveiling possible evolutionary mechanisms to enhance the kinetics of 2D protein crystallization in vivo.

biophysics↗

bioRxiv2019

Continuous, Topologically Guided Protein Crystallization Controls Bacterial Surface Layer Self-Assembly

Bacteria assemble the cell envelope using localized enzymes to account for growth and division of a topologically complicated surface1-3. However, a regulatory pathway has not been identified for assembly and maintenance of the surface layer (S-layer), a 2D crystalline protein coat surrounding the curved 3D surface of a variety of bacteria4,5. By specifically labeling, imaging, and tracking native and purified RsaA, the S-layer protein (SLP) from C. crescentus, we show that protein self-assembly alone is sufficient to assemble and maintain the S-layer in vivo. By monitoring the location of newly produced S-layer on the surface of living bacteria, we find that S-layer assembly occurs independently of the site of RsaA secretion and that localized production of new cell wall surface area alone is insufficient to explain S-layer assembly patterns. When the cell surface is devoid of a pre-existing S-layer, the location of S-layer assembly depends on the nucleation characteristics of SLP crystals, which grow by capturing RsaA molecules freely diffusing on the outer bacterial surface. Based on these observations, we propose a model of S-layer assembly whereby RsaA monomers are secreted randomly and diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated into growing 2D S-layer crystals. The complicated topology of the cell surface enables formation of defects, gaps, and grain boundaries within the S-layer lattice, thereby guiding the location of S-layer assembly without enzymatic assistance. This unsupervised mechanism poses unique challenges and advantages for designing treatments targeting cell surface structures or utilizing S-layers as self-assembling macromolecular nanomaterials. As an evolutionary driver, 2D protein self-assembly rationalizes the exceptional S-layer subunit sequence and species diversity6.

biophysics↗

bioRxiv2018

Retinoid X receptor alpha is a spatiotemporally-specific therapeutic target for doxorubicin-induced cardiomyopathy in adult zebrafish

While the genetic suppressor screen is efficient in suggesting therapeutic genes, this strategy has yet to be successful for cardiomyopathies in vertebrates. To develop such a strategy, we recently established a mutagenesis screen platform in zebrafish for systematic discovery of genetic modifiers of doxorubicin-induced cardiomyopathy (DIC). Here, we further revealed both molecular and cellular insights of the first salutary modifier emerged from the screen, i.e. gene-breaking transposon (GBT) 0419 that affects the retinoid X receptor alpha a (rxraa) gene. First, by rescuing the mutation in tissue-specific manner with multiple Cre-loxP systems, we demonstrated that the endothelial, but not myocardial or epicardial, function of rxraa is primary to this cardioprotective effects. Next, we showed that the rxraa-associated salutary effects on DIC were conferred partially by the activation of retinoid acid (RA) signaling. Finally, we identified isotretinoin and bexarotene, 2 US Food and Drug Administration-approved RXRA agonists that are effective in treating adult zebrafish DIC when administered during the early, but not the late, phase of DIC progression. Collectively, we provided the first in vivo genetic evidence in supporting RXRA as the therapeutic target for DIC, and uncovered a previously unrecognized spatiotemporally-restricted mechanism for this gene-based therapeutic strategy. Our study also justified that searching salutary modifiers via zebrafish mutagenesis screen can be effective in discovering new therapeutic targets for cardiomyopathies.

genetics↗