Biology subjects

Beis, K.

Publications and source records attributed to Beis, K..

13 recordsLinked to original sources

bioRxiv2026

Structural basis of polyspecific drug recognition by MRP2

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a hepatobiliary ATP-binding cassette transporter that mediates the efflux of endogenous conjugates and many therapeutic agents, yet the basis of its broad ligand specificity remains poorly understood. Here we combine cryo-electron microscopy, ATPase measurements and cell-based transport inhibition assays to define how MRP2 recognizes clinically relevant compounds from distinct chemical classes. We find that chemically diverse ligands occupy overlapping regions of a shared transmembrane cavity but adopt different poses, contact networks and stoichiometries. Ligand recognition is mediated predominantly by a permissive environment complemented by ligand-specific polar interactions, rather than by a single conserved pharmacophore. These findings establish a structural framework for MRP2 polyspecificity and provide insight into hepatobiliary drug disposition and transporter-mediated drug-drug interactions.

biochemistry↗

bioRxiv2026

An Automated HDX-MS Platform for in situ characterisation of Membrane Proteins

The structural study of membrane proteins has traditionally relied on detergent-based extraction from cellular membranes. Although native-like reconstitution approaches have advanced, fully understanding membrane protein dynamics requires examining them within their native membrane environment. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for probing structural dynamics in reconstituted systems, but the presence of the lipid bilayer introduces considerable complexity, limiting broader adoption under physiological conditions. Here, we present the first fully automated HDX-MS platform incorporating a two-stage delipidation workflow. We applied this approach to monitor the dynamics of the ABC transporter MsbA in isolated inner membrane vesicles (IIMVs) from Escherichia coli through its ATPase cycle. IIMVs revealed distinct dynamic features within the nucleotide binding domains and substrate binding cavity, highlighting physiologically relevant motions not observed with detergent solubilised MsbA. This platform significantly advances HDX-MS and underscores the importance of studying membrane proteins in native lipid environments.

biochemistry↗

bioRxiv2025

TraN variants mediate conjugation species specificity of IncA/C, IncH and Acinetobacter baumannii plasmids

IncA/C and IncH plasmids commonly carry antimicrobial resistance genes, notably blaNDM-1. Although these plasmids disseminate among Gram-negative pathogens via conjugation, the mechanisms underlying mating pair stabilisation (MPS) and conjugation species specificity remain poorly understood. In IncF plasmids, MPS is mediated by interactions between outer membrane proteins (OMP) encoded by the plasmids in the donor (TraN) and by the chromosome in the recipient. Using the Plascad database, we extracted 1,436 TraN sequences: 62.5% (898/1,436), mainly in IncF plasmids, are 550-660aa (we renamed TraN short, TraNS); 15% (216/1,436), in IncA/C plasmids, are 880-950aa (TraN medium, TraNM); and 11% (160/1,436), in IncH plasmids, are 1,050-1,070aa (TraN long, TraNL). A group of six TraN from Acinetobacter baumannii plasmids (891aa) were designated TraN V-shaped (TraNV). Like TraNS, TraNM and TraNL contain base and distal tip domains essential for conjugation, whereas TraNV has a base and two distinct tip domains forming a V-shaped structure. TraNM, TraNL and TraNV determine conjugation species specificity, with TraNL cooperating with OmpA. Tip swapping reverses conjugation specificity, revealing how TraNM and TraNL diversity influence plasmid host range and AMR dissemination. Our new data reveal the molecular basis of plasmid host specificity and broaden our understanding of how conjugation drives the dissemination of antimicrobial resistance genes among clinically relevant bacteria.

microbiology↗

bioRxiv2025

The BacA(SbmA) Importer of Symbiotically Important Legume Nodule Cysteine-Rich Peptides: Insights into Protein Architecture, Function, and Evolutionary Implications

Some legumes encode families of NCR (Nodule-Cysteine-Rich) peptides that cause their rhizobial partners to terminally differentiate during the development of a nitrogen-fixing symbiosis. Sinorhizobium meliloti, whose plant hosts Medicago truncatula and M. sativa express ca. 600 NCR peptides during root nodule development, possesses a symbiotically essential BacASm protein that imports certain NCR peptides into the cytoplasm. This import permits proteolytic degradation of the NCR peptides, thereby protecting the endocytosed bacteria from their antimicrobial peptide-like lethality, while also allowing certain NCR peptides to undergo their symbiotically critical interactions with cytoplasmic components, for example heme-sequestration in the case of NCR247. BacAs Escherichia coli ortholog SbmAEc can restore a wildtype phenotype to a{Delta} bacASm mutant. Our study employed 54 S. meliloti bacASm missense mutants (35 to cysteine and 19 to glycine) that we tested for protein production, ability to establish a nitrogen-fixing symbiosis, and their susceptibility to killing by higher levels of the NCR247 and the Bac7(1-35) peptides. We also used the Single Cysteine Accessibility Method to make topological inferences. Our detailed genetic, biochemical, structural, and physiological analyses have revealed that BacASm and SbmA homodimers function as finely tuned import machines, whose structures can be relatively easily disrupted by single amino acid changes. Our discovery that several mutations that differentially separate nitrogen-fixation, NCR247 import, and Bac7(1-35) import map to the lining of the peptide-binding cavity in the outward-open SbmA/BacA conformation suggests a molecular explanation the other otherwise paradoxical observation that SbmA/BacAs from pathogens can fully replace BacASm, whereas BacAs from other rhizobia cannot. Significance StatementSinorhizobium meliloti BacASm and Escherichia coli SbmAEc are closely related proteins that function as homodimeric transporters to import peptides and other cargos through the cytoplasmic membrane into the cytoplasm. BacA is critical for S. meliloti to establish a nitrogen-fixing symbiosis with its legume hosts because of its ability to import Nodule Cysteine-Rich (NCR) nodule-specific plant peptides. This import protects the bacteria inside the nodule from the potentially lethal effects of these NCR peptides while also enabling NCRs to make their intracellular interactions that are necessary for symbiosis. Our extensive multidisciplinary studies offer new insights into function of BacA/SbmA transporters and provide a molecular explanation for why BacA/SbmA orthologs from mammalian pathogens can replace BacASm but those from other rhizobia cannot.

biochemistry↗

bioRxiv2025

R27 pilus biogenesis is promiscuous and electrostatic perturbations impair functionality

During conjugation, plasmid DNA is transferred from donor to recipient bacteria via the plasmid-encoded mating pilus, formed as thin helical assemblies of polymerised pilin subunits. In the IncHI1 R27 plasmid-encoded pilus, the TrhA pilin undergoes cyclisation (via a peptide bond between Gly1 and Asp69), essential for conjugation. Gly1 and Asp69 are exposed on the pilus surface and conserved in all TrhA pilins in the Plascad database. Substituting Asp69 with Asn, Ala, Gly, or Arg did not prevent cyclisation or pilus formation, which remained structurally indistinguishable from the wild type. Conjugation efficiency of the Asp69 substitutions across multiple recipient species correlated with side chain size, in the order Asp69Asn > Asp69Ala > Asp69Gly. However, Asp69Arg, as well as Asp69Lys and Gly1Lys substitutions abolished conjugation, likely due to the positively charged pilus surface (opposite to the wild-type negative charge) forming unfavourable electrostatic interactions with the recipient outer membranes inner leaflet, composed solely of zwitterionic phosphatidylethanolamine (PE). Consistently, conjugation was rescued in recipients lacking PE. These findings indicate strong selective pressure to maintain Gly1 and Asp69, as efficient DNA transfer depends on precise electrostatic and steric constraints of the pilus surface.

microbiology↗

bioRxiv2025

Phospholipid-independent biogenesis of a functional RP4 conjugation pilus

Conjugation, the process of DNA transfer between bacteria, is initiated universally by the formation of a mating pair formation (MPF) via a conjugative pilus. Conjugation of the IncP RP4 plasmid is mediated by short, non-retractable, rigid mating pili. Here, we report the cryo-EM structure of the RP4 pilus at 2.75 [A] resolution. Uniquely, and consistently with quantitative mass spectral analysis, this revealed that the cyclic TrbC pilin subunit is not lipidated. Consistently, an E. coli pgsA mutant lacking phosphatidylglycerol (PG) can serve as a donor of RP4 but not of R27, encoding the H-pilus consisting of PG-associated cyclic pilin subunits (TrhA). The RP4 is the first example of a lipid-independent functional mating pilus. This discovery not only challenges the long-held assumption that an amphipathic lipid moiety is essential for the construction of conjugative pili and for MPF, but also expanding our understanding of the diverse mechanisms that bacteria employ to transfer genetic material.

microbiology↗

bioRxiv2024

Cryo-EM structure of the conjugation H-pilus reveals the cyclic nature of the TrhA pilin

Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F-pilus, are dynamic tubular structures, composed of a polymerized pilin, that mediate the initial donor-recipient interactions, a process known as mating pair formation (MPF). IncH are low-copy-number plasmids, traditionally considered broad host range, which are found in bacteria infecting both humans and animals. The reference IncHI1 plasmid R27, isolated from Salmonella enterica serovar Typhi, encodes the conjugative H-pilus subunit TrhA containing 74 residues after cleavage of the signal sequence. Here, we show that the H-pilus forms long filamentous structures that mediate MPF, and describe its cryo electron-microscopic (cryo-EM) structure at 2.2 [A] resolution. Like the F pilus, the H-pilin subunits form helical assemblies with phospholipid molecules at a stochiometric ratio of 1:1. While there were previous reports that the T-pilus from Agrobacterium tumefaciens was composed of cyclic subunits, three recent cryo-EM structures of the T-pilus found no such cyclization. Here, we report that the H-pilin is cyclic, with a covalent bond connecting the peptide backbone between the N- and C-termini. Both the cryo-EM map and mass spectrometry revealed cleavage of the last five residues of the pilin, followed by cyclization via condensation of the amine and carboxylate residues. The cyclic nature of the pilin could stabilize the pilus and may explain the high incidence of IncH plasmid dissemination. SignificanceA major medical challenge is the spread of bacteria which are resistant to antibiotics. The resistance genes are spread via mobilized DNA, mainly via a process named conjugation. During conjugation, a resistant bacterium (donor), transfers the resistance DNA to another bacterium (recipient) in a contact-dependent manner. The initial donor-recipient interaction is mediated by a hollow filament expressed by the donor, named the conjugation pilus, that binds the recipient. This pilus is built via polymerization of a small protein subunit, pilin. Here, we report the atomic structure of the H-pilus, whose pilin subunit has an unusual cyclic structure where the N- and C-termini of the protein are covalently linked by a peptide bond.

microbiology↗

bioRxiv2024

Cryo-EM structure and evolutionary history of the conjugation surface exclusion protein TraT

Conjugation plays a major role in dissemination of antimicrobial resistance genes. Following transfer of IncF-like plasmids, recipients become refractory to a second wave of conjugation with the same plasmid via entry (TraS) and surface (TraT) exclusion mechanisms. Here, we show that TraT from the pKpQIL and F plasmids (TraTpKpQIL and TraTF) exhibits plasmid surface exclusion specificity. The cryo-EM structures of TraTpKpQIL and TraTF revealed that they oligomerise into decameric champagne bottle cork-like structures, which are anchored to the outer membrane via a diacylglycerol modified -helical barrel domain. Unexpectedly, we identified chromosomal TraT homologues from multiple Gram-negative phyla which formed numerous deep-branching lineages in a phylogenetic tree of TraT sequences. Plasmid-associated TraT sequences largely cluster into two separate lineages that have more recently evolved, incorporating TraT from Enterobacterales IncF and Legionellaceae F-like plasmids. These findings suggest that different plasmid backbones have acquired and co-opted TraT on independent occasions.

microbiology↗

bioRxiv2023

The interaction of the F-like plasmid-encoded TraN isoforms with their cognate outer membrane receptors

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of seven TraN sequence types, which are grouped into four structural types, we named TraN, TraN{beta}, TraN{gamma}, TraN{delta}. Moreover, we have shown specific pairing between TraN and OmpW, TraN{beta} and OmpK36 of Klebsiella pneumoniae, TraN{gamma} and OmpA and TraN{delta} and OmpF. In this study we found that although structurally similar, TraN encoded by the pSLT plasmid (TraN2) binds OmpW in both Escherichia coli and Citrobacter rodentium while TraN encoded by the R100-1 plasmid (TraN1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraN{beta}-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraN{beta} can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. ImportanceConjugation plays a central role in the spread of antimicrobial resistance genes amongst bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilisation (MPS), mediated by tight interactions between the plasmid encoded outer membrane protein (OMP) TraN in the donor (of which there are seven sequence types grouped into the four structural isoforms , {beta}, {gamma}, {delta}) and an OMP receptor in the recipient (OmpW, OmpK36, OmpA and OmpF, respectively). In this study we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.

microbiology↗

bioRxiv2022

The antibiotic phazolicin displays a dual mode of uptake in Gram-negative bacteria

Phazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer Rhizobium sp. Pop5. Using genetic and biochemical techniques, we here identified BacA and YejABEF as two importers of PHZ in a sensitive model strain Sinorhizobium meliloti Sm1021. BacA and YejABEF are members of SLiPT and ABC transporter families of non-specific peptide importers, respectively. The uptake of PHZ by two distinct families of transporters dramatically decreases the naturally occurring rate of resistance. Moreover, since both BacA and YejABEF are essential for the development of functional symbiosis of rhizobia with leguminous plants, the acquisition of PHZ resistance via the inactivation of transporters is further disfavoured since single bacA or yejABEF mutants are unable to propagate in root nodules. Crystal structures of the periplasmic subunit YejA from S. meliloti and Escherichia coli revealed fortuitous bound peptides, suggesting a non-specific peptide-binding mechanism that facilitates the uptake of PHZ and other antimicrobial peptides. SIGNIFICANCEMany bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides kill bacteria by either membrane disruption or inhibiting essential intracellular processes. The Achilles heel of the latter type of antimicrobials is their dependence on transporters to enter the susceptible bacteria since mutations in such transporters result in resistance. We describe here how the ribosome-targeting peptide phazolicin, produced by Rhizobium sp. Pop5, uses two different transporters, BacA and YejABEF, to get into the cells of the symbiotic bacterium Sinorhizobium meliloti. This dramatically reduces the probability of resistance acquisition. Both transporters need to be inactivated for phazolicin resistance acquisition. Since these transporters are also crucial in S. meliloti for its symbiotic association with host plants, their inactivation in biological settings is highly unlikely. This makes PHZ an attractive lead for the development of a biocontrol agent with potential for use in agriculture.

molecular biology↗

bioRxiv2022

Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. In a murine pneumonia model, KP mutants harbouring L3 insertions have a competitive disadvantage relative to a strain expressing wild-type OmpK36 in the absence of antibiotics. This explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies. Author summaryRapidly rising rates of antibiotic resistance among Klebsiella pneumoniae (KP) necessitate a comprehensive understanding of the diversity, spread and clinical impact of resistance mutations. In KP, mutations in outer membrane porins play an important role in mediating resistance to carbapenems, a key class of antibiotics. Here we show that resistance mutations in the extracellular loop 3 (L3) region of the OmpK36 porin are found at high prevalence among clinical genomes and we characterise their diversity and impact on resistance and virulence. They include amino acid insertions of Aspartate (D), Glycine-Aspartate (GD) and Threonine-Aspartate (TD), which act by decreasing the pore size and restricting entry of carbapenems into the bacterial cell. We show that these L3 insertions are associated with large clonal expansions of resistant lineages and impose only a low fitness cost. Critically, strains harbouring L3 insertions remain susceptible to novel drugs, including beta lactam-beta lactamase inhibitor combinations. This study highlights the importance of monitoring the emergence and spread of strains with OmpK36 L3 insertions for the control of resistant KP infections and provides crucial data for drug development and treatment strategies.

microbiology↗

bioRxiv2020

OmpK36 and TraN facilitate conjugal transfer of the Klebsiella pneumoniae carbapenem resistance plasmid pKpQIL

We investigated the mechanism of conjugal transfer of the endemic Klebsiella pneumoniae carbapenem resistance plasmid, pKpQIL. Transfer efficiency of this plasmid was found to be dependent on the expression of the major outer membrane porin, OmpK36, in recipient cells. We also found that conjugal uptake is reduced in recipients expressing an OmpK36 isoform associated with the globally pervasive K. pneumoniae ST258 clade (OmpK36ST258). This reduction was attributed to a glycine-aspartate insertion in loop 3 of OmpK36ST258, which constricts the pore by 26%. Deletion of finO, which encodes an RNA-binding protein, derepressed transfer of pKpQIL and enabled visualisation of the conjugation pilus and OmpK36-dependent conjugation in real time. While deletion of traN abolished pKpQIL conjugation, substituting traN in pKpQIL with its homologue from R100-1 circumvented OmpK36 dependency. These results suggest that OmpK36 in recipient K. pneumoniae and the pKpQIL-encoded TraN in donor bacteria cooperate to facilitate plasmid transfer. This is the first report since 1998 to suggest a novel recipient cell receptor for IncF plasmid transfer and supports the idea that TraN mediates receptor specificity for plasmids belonging to this incompatibility group.

microbiology↗

bioRxiv2018

Solid immersion microscopy readily and inexpensively enables 12 nm resolution on plunge-frozen cells

Super-resolution fluorescence microscopy achieves 20-30 nm resolution by using liquid-immersion objectives to optimize light collection and chemical sample fixation to minimize image blurring. It is known that fluorophore brightness increases substantially under cryogenic conditions and that cryo-fixation is far superior in preserving ultrastructure. However, cryogenic conditions have not been exploited to improve resolution or sample quality because liquid immersion media freezes at the objective, losing its optical properties. Here, simply by replacing the immersion fluid with a low-cost super-hemispherical solid immersion lens (superSIL), we effortlessly achieve <8 nm localisation precision and 12 nm resolution under cryogenic conditions in a low-cost, low-tech system. This is to our knowledge the best resolution yet attained in biological samples. Furthermore, we demonstrate multicolour imaging and show that the inexpensive setup outperforms 10-fold more costly super-resolution microscopes. By also removing the barrier to total internal reflection fluorescence imaging of mammalian cells under cryogenic conditions, superSIL microscopy delivers a straightforward route to achieve unmatched nanoscale resolution on both bacterial and mammalian cell samples, which any laboratory can effortlessly and inexpensively implement.

biophysics↗