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Conjugative transfer through type IV secretion multiprotein complexes is the most important means of spreading antimicrobial resistance. Plasmid pIP501, frequently found in clinical Enterococcus faecalis and Enterococcus faecium isolates, is the first Gram-positive (G+) conjugative plasmid for which self-transfer to Gram-negative (G−) bacteria has been demonstrated. The pIP501-encoded type IV secretion system (T4SS) protein TraN localizes to the cytoplasm and shows specific DNA binding. The specific DNA-binding site upstream of the pIP501 origin of transfer ( oriT) was identified by a novel footprinting technique based on exonuclease digestion and sequencing, suggesting TraN to be an accessory protein of the pIP501 relaxase TraA. The structure of TraN was determined to 1.35 Å resolution. It revealed an internal dimer fold with antiparallel β-sheets in the centre and a helix-turn-helix (HTH) motif at both ends. Surprisingly, structurally related proteins (excisionases from T4SSs of G+ conjugative transposons and transcriptional regulators of the MerR family) resembling only one half of TraN were found. Thus, TraN may be involved in the early steps of pIP501 transfer, possibly triggering pIP501 TraA relaxase activity by recruiting the relaxosome to the assembled mating pore. [ABSTRACT FROM AUTHOR]

Conjugative plasmid transfer presents a serious threat to human health as the most important means of spreading antibiotic resistance and virulence genes among bacteria. The required direct cell-cell contact is established by a multi-protein complex, the conjugative type IV secretion system (T4SS). The conjugative core complex spans the cellular envelope and serves as a channel for macromolecular secretion. T4SSs of Gram-negative (G-) origin have been studied in great detail. In contrast, T4SSs of Gram-positive (G+) bacteria have only received little attention thus far, despite the medical relevance of numerous G+ pathogens (e.g. enterococci, staphylococci and streptococci). This study provides structural information on the type IV secretion (T4S) protein TraKΔ of the G+ broad host range Enterococcus conjugative plasmid pIP501. The crystal structure of the N-terminally truncated construct TraKA was determined to 3.0 Å resolution and exhibits a novel fold. Immunolocalization demonstrated that the protein localizes to the cell wall facing towards the cell exterior, but does not exhibit surface accessibility. Circular dichroism, dynamic light scattering and size-exclusion chromatography confirmed the protein to be a monomer. With the exception of proteins from closely related T4SSs, no significant sequence or structural relatives were found. This observation marks the protein as a very exclusive, specialized member of the pIP501 T4SS. [ABSTRACT FROM AUTHOR]

Highlights: [•] The conjugative transfer mechanism of broad-host-range, Enterococcus sex pheromone and Clostridium plasmids is reviewed. [•] Comparisons with Gram-negative type IV secretion systems are presented. [•] The current understanding of the unique Streptomyces double stranded DNA transfer mechanism is reviewed. [Copyright &y& Elsevier]

Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gramnegative (G-) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G-) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G- bacteria, we propose a new classification scheme of VirB8-like proteins. [ABSTRACT FROM AUTHOR]

The universally conserved protein YidC aids in the insertion and folding of transmembrane polypeptides. Supposedly, a charged arginine faces its hydrophobic lipid core, facilitating polypeptide sliding along YidC's surface. How the membrane barrier to other molecules may be maintained is unclear. Here, we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon ribosome or ribosome-nascent chain complex (RNC) binding. In contrast to monomeric YidC structures, an AlphaFold parallel YidC dimer model harbors a pore. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, single-molecule fluorescence photobleaching observations, and crosslinking experiments. In the dimeric model, the conserved arginine and other residues interacting with nascent chains point into the putative pore. This result suggests the possibility of a YidC-assisted insertion mode alternative to the insertase mechanism. [ABSTRACT FROM AUTHOR]

Biological ion-selective channels possess an unparalleled combination of high ion conductance and selectivity. Pores with comparable characteristics are highly required in industrial applications, e.g., for ion separation from brines. Yet, bio-channels' application is limited due to their instability, complexity in production, and low surface density. Artificial channels with a satisfactory trade-off between selectivity and conductance may represent an alternative. We propose taking advantage of the amphiphilic properties and high chemical variability of polyhedral oligomeric silsesquioxane (POSS), thus far used as a filler in nano-composite membranes. We designed and synthesized diblock copolymers where the hydrophobic block contained POSS cages decorated with isobutyl groups. Polymers containing four (SP5B3-5) or eight (Au22) POSS cages appeared capable of self-assembling into alkali ion-selective pores in planar lipid bilayers. Based on polymer structure and dose effect experiments, we propose that SP5B3-5 forms a tetrameric pore, with two monomers from each membrane leaflet, and Au22 forms an antiparallel dimeric pore. Both polymers prefer cations over anions. The unitary conductance decreases in the order Cs+ > K+ > Na+ of the Eisenman sequence I, indicating that the selectivity primarily stems from the penalty for ion dehydration. The permeabilities for Cs+, K+ and Na+ relate as 2.5 : 1.9 : 1 for SP5B3-5, and as 2.5 : 1.5 : 1 for Au22. The observation is compatible with a conductivity pathway where six oxygen atoms of the surrounding four POSS cages coordinate an ion within the pore. The artificial POSS-based pores pave the way for producing ion-selective membranes with high throughput. [Display omitted] • A set of amphiphilic diblock copolymers with POSS-containing hydrophobic blocks was checked on the ability to form ion-conductive pores. • Two of the polymers, SP5B3-5 and Au22, could self-assemble into cation-selective pores. • Both polymers form pores with different selectivity to monovalent cations, seemingly based on the ion dehydration penalty. • We propose that within the pore, ions are stabilized by oxygens of POSS cages. • The novel artificial channels have selectivity and conductance comparable to biological ion channels. [ABSTRACT FROM AUTHOR]

Viral inhibitors, such as pleconaril and vapendavir, target conserved regions in the capsids of rhinoviruses (RVs) and enteroviruses (EVs) by binding to a hydrophobic pocket in viral capsid protein 1 (VP1). In resistant RVs and EVs, bulky residues in this pocket prevent their binding. However, recently developed pyrazolopyrimidines inhibit pleconaril-resistant RVs and EVs, and computational modeling has suggested that they also bind to the hydrophobic pocket in VP1. We studied the mechanism of inhibition of pleconaril-resistant RVs using RV-B5 (1 of the 7 naturally pleconaril-resistant rhinoviruses) and OBR-5-340, a bioavailable pyrazolopyrimidine with proven in vivo activity, and determined the 3D-structure of the protein-ligand complex to 3.6 Å with cryoelectron microscopy. Our data indicate that, similar to other capsid binders, OBR-5-340 induces thermostability and inhibits viral adsorption and uncoating. However, we found that OBR-5-340 attaches closer to the entrance of the pocket than most other capsid binders, whose viral complexes have been studied so far, showing only marginal overlaps of the attachment sites. Comparing the experimentally determined 3D structure with the control, RV-B5 incubated with solvent only and determined to 3.2 Å, revealed no gross conformational changes upon OBR-5-340 binding. The pocket of the naturally OBR-5-340-resistant RV-A89 likewise incubated with OBR-5-340 and solved to 2.9 Å was empty. Pyrazolopyrimidines have a rigid molecular scaffold and may thus be less affected by a loss of entropy upon binding. They interact with lessconserved regions than known capsid binders. Overall, pyrazolopyrimidines could be more suitable for the development of new, broadly active inhibitors. [ABSTRACT FROM AUTHOR]

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecafis and Enterococcus cium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for piP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring piP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGATMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchito- hexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and b degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosyla Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope. [ABSTRACT FROM AUTHOR]