Biology subjects

ter Beek, J.

Publications and source records attributed to ter Beek, J..

8 recordsLinked to original sources

bioRxiv2025

Pili are essential for conjugation also in many Gram-positive bacteria

Type IV secretion systems (T4SS) enable the spread of antibiotic resistance and other virulence factors. In Gram-positive bacteria, T4SSs have long been thought to lack VirB2-like proteins that form conjugative pili and instead rely on adhesins for cell-cell contacts. Yet, it has remained unclear how subsequent DNA transfer (conjugation) is physically mediated. Here we identify a VirB2-like protein, PrgFB2, from the clinically isolated conjugative plasmid pCF10 in Enteroccocus faecalis and show that it is essential for conjugation. Structural modeling confidently predicts a pilus-like assembly for PrgFB2. We validate this prediction through mutagenesis, conjugation assays, and targeted chemical labeling. By combining various machine learning bioinformatic techniques, we analysed >1000 Gram-positive conjugative plasmids from diverse species, including major pathogens, and identified pili forming VirB2-like proteins in almost all of them. Our findings overturn the prevailing view that Gram-positive T4SSs lack pili. This discovery provides a new framework for understanding horizontal gene transfer and highlights critical targets for combating antimicrobial resistance and virulence in Gram-positive bacteria.

microbiology↗

bioRxiv2024

Breaking Barriers: pCF10 Type 4 Secretion System relies on a self-regulating muramidase to modulate the cell wall

Conjugative Type 4 Secretion Systems (T4SS) are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient mating bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has 3 predicted extracellular hydrolase domains, LytM, SLT and CHAP. Here, we report the structure of the LytM domain, and show that its active site is degenerate and lacks the active site metal. Further, we show that only the predicted SLT domain is functional, and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Further, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information towards understanding the function of Gram-positive T4SSs.

biochemistry↗

bioRxiv2023

A distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha and Betaproteobacteria

Most bacteria are surrounded by an essential protective mesh-like structure called peptidoglycan, made of glycan chains crosslinked through short peptides by enzymes known as transpeptidases. Of these, penicillin-binding DD-transpeptidases connect adjacent peptide stems between their 4th and 3rd amino acids (4,3-type), D-alanine and a meso-diaminopimelic acid (mDAP) in Gram negatives, whereas LD-transpeptidases make the 3,3-type between mDAP3 residues. While these two processes explain the formation of crosslinks in most bacteria, recent investigations involving non-model species have brought to light novel crosslinking mechanisms that point to the existence of less-explored groups of peptidoglycan crosslinking enzymes. Here, we present the identification and characterization of a novel LD-transpeptidase found in the acetic acid bacterial Gluconobacter oxydans, named LDTGo, which performs 1,3-type crosslinks between L-Ala1 and mDAP3. LDTGo-like proteins are conserved among Alpha and Betaproteobacteria species that do not encode LD3,3-transpeptidases. Using a highly active ortholog, we demonstrated in vitro that this enzyme can work with non-terminal peptide bonds in the crosslinking process. This property is different from the strict specificity of typical LD- and DD-transpeptidases, which only deal with terminal peptide bonds. The high-resolution crystal structure of LDTGo revealed significant distinctions when compared to 3,3-type LD-transpeptidases. These include a proline-rich region near the N-terminus that restricts substrate access to the active site, and an unprecedented cavity designed to accommodate both the glycan chain and the peptide stem from donor muropeptides, a feature that exhibits broad conservation among LD1,3-transpeptidases. Finally, we demonstrated the involvement of DD-crosslinking turnover in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between structurally distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans.

microbiology↗

bioRxiv2023

In vitro reconstitution reveals membrane clustering and double-stranded RNA recruitment by the enteroviral AAA+ ATPase 2C

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2Cs association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divides the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

microbiology↗

bioRxiv2022

Structural foundation for the role of enterococcal PrgB in conjugation, biofilm formation and virulence

Type 4 Secretion Systems are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. In Gram-positives, these secretion systems often rely on surface adhesins to enhance cellular aggregation and mating pair formation. One of the best studied adhesins is PrgB from the conjugative plasmid pCF10 of Enterococcus faecalis, which has been shown to play major roles in conjugation, biofilm formation and importantly also in bacterial virulence. Since prgB orthologs exist on a large number of conjugative plasmids in various different species, this makes PrgB a model protein for this widespread virulence factor. After characterizing the polymer adhesin domain of PrgB previously, we here report the structure for almost the entire remainder of PrgB, which reveals that PrgB contains four immunoglobulin-like domains. Based on this new insight, we re-evaluate previously studied variants and present new in vivo data where specific domains or conserved residues have been removed. For the first time, we can show a decoupling of cellular aggregation from biofilm formation and conjugation in prgB mutant phenotypes. Based on the presented data, we propose a new functional model to explain how PrgB mediates its different functions. We hypothesize that the Ig-like domains act as a rigid stalk that presents the polymer adhesin domain at the right distance from the cell wall.

biochemistry↗

bioRxiv2022

Structural and functional characterization of TraI from pKM101 reveals basis for DNA processing

Type 4 Secretion Systems (T4SSs) are large and versatile protein machineries that facilitate the spread of antibiotic resistance and other virulence factors via horizontal gene transfer. Conjugative T4SSs depend on relaxases to process the DNA in preparation for transport. TraI from the well-studied conjugative plasmid pKM101 is one such relaxase. Here, we report the crystal structure of the trans-esterase domain of TraI in complex with its substrate oriT DNA, highlighting the conserved DNA binding mechanism of conjugative relaxases. Additionally, we present an apo structure of the trans-esterase domain of TraI that includes most of the flexible thumb region. This allows us for the first time to visualize the large conformational change of the thumb domain upon DNA binding. We also characterize the DNA binding, nicking and religation activity of the trans-esterase domain, helicase domain and full-length TraI. Unlike previous indications in the literature, our results reveal that the TraI trans-esterase domain from pKM101 behaves in a conserved manner with its homologs from the R388 and F plasmids.

biochemistry↗

bioRxiv2020

Enterococcal PrgU mitigates PrgB overexpression toxicity by binding to intergenic RNA downstream of the PQ promoter

Efficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the sex pheromone cCF10, which induces the expression of the Type 4 Secretion System (T4SS) genes controlled by the PQ promoter. The pheromone responsive PQ promoter is strictly regulated to prevent overproduction of the prgQ operon, which contains the T4SS, and to limit the cell toxicity caused by overproduction of PrgB, a T4SS adhesin involved in cellular aggregation. PrgU plays an important role in regulating this toxicity by decreasing PrgB production. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. With a combination of lacZ reporter fusion, northern blot, and RNAseq analyses, we provide evidence that PrgU binds a specific RNA sequence within the intergenic region (IGR), ca 400 bp downstream of the PQ promoter. PrgU-IGR binding reduces levels of downstream transcripts, with the strongest decrease seen for prgB messages. Consistent with these findings, we determined that pCF10-carrying cells expressing prgU decreased transcript levels more rapidly than isogenic cells deleted of prgU. Finally, purified PrgU bound RNA in vitro, but without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors to bind its RNA target in vivo. Together, our results support a working model where PrgU binding to the IGR serves to recruit RNase(s) for targeted degradation of downstream transcripts.Importance Bacteria utilize Type 4 Secretion Systems (T4SS) to efficiently transfer DNA from donor to recipient cells, thereby spreading genes encoding for antibiotic resistance as well as various virulence factors. The conjugative plasmid pCF10 from Enterococcus faecalis, originally isolated from clinical isolates, serves as a model system for these processes in Gram-positive bacteria. It is very important to strictly regulate the expression of the T4SS proteins for the bacteria, as some of these proteins are highly toxic to the cell. Here, we identify the mechanism by which PrgU performs its delicate fine tuning of the expression levels. As prgU genes are present in various conjugative plasmids and transposons, this provides an important new insight into the bacterial repertoire of regulation mechanisms of these clinically important systems.View Full Text

molecular biology↗

bioRxiv2020

Enterococcal PrgA extends far outside the cell and provides surface exclusion to protect against unwanted conjugation

Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via Type 4 Secretion Systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a 2nd copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggest that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.

biochemistry↗