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The bacterial cell-wall peptidoglycan is made of glycan strands crosslinked by short peptide stems. Crosslinks are catalyzed by DD-transpeptidases (4,3-crosslinks) and LD-transpeptidases (3,3-crosslinks). However, recent research on non-model species has revealed novel crosslink types, suggesting the existence of uncharacterized enzymes. Here, we identify an LD-transpeptidase, LDT_Go, that generates 1,3-crosslinks in the acetic-acid bacterium Gluconobacter oxydans. LDT_Go-like proteins are found in Alpha- and Betaproteobacteria lacking LD3,3-transpeptidases. In contrast with the strict specificity of typical LD- and DD-transpeptidases, LDT_Go can use non-terminal amino acid moieties for crosslinking. A high-resolution crystal structure of LDT_Go reveals unique features when compared to LD3,3-transpeptidases, including a proline-rich region that appears to limit substrate access, and a cavity accommodating both glycan chain and peptide stem from donor muropeptides. Finally, we show that DD-crosslink turnover is involved in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans.

Conjugative type 4 secretion systems (T4SSs) are the 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 bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has three predicted extracellular hydrolase domains: metallo-peptidase (LytM), soluble lytic transglycosylase (SLT), and cysteine, histidine-dependent amidohydrolases/peptidases (CHAP). Here, we report the structure of the LytM domain and show that its active site is degenerate and lacks the active site metal. Furthermore, we show that only the predicted SLT domain is functional in vitro 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. Furthermore, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information for understanding the function of Gram-positive T4SSs.

IMPORTANCE: Antibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from Enterococcus faecalis. This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in E. faecalis as well as other medically relevant Gram-positive bacteria. Antibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from Enterococcus faecalis. This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in E. faecalis as well as other medically relevant Gram-positive bacteria.

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 2C’s 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.

To explore favourable niches while avoiding threats, many bacteria use a chemotaxis navigation system. Despite decades of studies on chemotaxis, most signals and sensory proteins are still unknown. Many bacterial species release d-amino acids to the environment; however, their function remains largely unrecognized. Here we reveal that d-arginine and d-lysine are chemotactic repellent signals for the cholera pathogen Vibrio cholerae. These d-amino acids are sensed by a single chemoreceptor MCPDRK co-transcribed with the racemase enzyme that synthesizes them under the control of the stress-response sigma factor RpoS. Structural characterization of this chemoreceptor bound to either d-arginine or d-lysine allowed us to pinpoint the residues defining its specificity. Interestingly, the specificity for these d-amino acids appears to be restricted to those MCPDRK orthologues transcriptionally linked to the racemase. Our results suggest that d-amino acids can shape the biodiversity and structure of complex microbial communities under adverse conditions.

Type 4 secretion systems are large and versatile protein machineries that facilitate the spread of antibiotic resistance and other virulence factors via horizontal gene transfer. Conjugative type 4 secretion systems 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. In addition, 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 subdomain 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.

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 (Ig)-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.

Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2_CORE, are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2_COREexo^–, was used to limit DNA degradation. While no redox signal is apparent for Pol2_COREexo^– on DNA-modified electrodes, a large cathodic signal centered at −140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2_COREexo^–, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]²⁺, to oxidized Pol2_COREexo^– bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIII^Y82A, a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2_COREexo^–. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2_COREexo^– via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

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 second 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 suggests 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. 

The most frequently recurring cancer-associated DNA polymerase epsilon (Pol epsilon) mutation is a P286R substitution in the exonuclease domain. While originally proposed to increase genome instability by disrupting exonucleolytic proofreading, the P286R variant was later found to be significantly more pathogenic than Pol epsilon proofreading deficiency per se. The mechanisms underlying its stronger impact remained unclear. Here we report the crystal structure of the yeast orthologue, Pol epsilon-P301R, complexed with DNA and an incoming dNTP. Structural changes in the protein are confined to the exonuclease domain, with R301 pointing towards the exonuclease site. Molecular dynamics simulations suggest that R301 interferes with DNA binding to the exonuclease site, an outcome not observed with the exonuclease-inactive Pol epsilon-D290A, E292A variant lacking the catalytic residues. These results reveal a distinct mechanism of exonuclease inactivation by the P301R substitution and a likely basis for its dramatically higher mutagenic and tumorigenic effects.

DNA polymerase ϵ (Pol ϵ), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2_CORE) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2_CORE (with the CysX cysteine motif) are likely to coordinate an Fe–S cluster. Here, we present two new crystal structures of Pol2_CORE with an Fe–S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol ϵ, Pol ϵ CysA_MUT (C2111S/C2133S), and Pol ϵ CysB_MUT (C2167S/C2181S) all have an Fe–S cluster that is not present in Pol ϵ CysX_MUT (C665S/C668S). Pol ϵ CysA_MUT and Pol ϵ CysB_MUT behave similarly to wild-type Pol ϵ in in vitro assays, but Pol ϵ CysX_MUT has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysX_MUT are inviable. In conclusion, Pol ϵ has a single Fe–S cluster bound at the base of the P-domain, and this Fe–S cluster is essential for cell viability and polymerase activity.