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Despite recent advances in our understanding of the structure and function of conjugative Type 4 Secretion Systems (T4SSs), there is still only very scarce data available for the ones from Gram-positive (G+) bacteria. This is a problem, as conjugative T4SSs are main drivers for the spread of antibiotic resistance genes and virulence factors. Here, we aim to increase our understanding of G+ systems, by using bioinformatic approaches to identify proteins that are conserved in all conjugative T4SS machineries and reviewing the current knowledge available for these components. We then combine this information with the most recent advances in structure prediction technologies to propose a structural model for a G+ T4SS from the model system encoded on pCF10. By doing so, we show that conjugative G+ T4SSs likely have more in common with their Gram-negative counterparts than previously expected, and we highlight the potential of predicted structural models to serve as a starting point for experimental design.

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.

Conjugative dissemination of mobile genetic elements (MGEs) among bacteria is initiated by assembly of the relaxosome at the MGE's origin-of-transfer (oriT) sequence. A critical but poorly defined step of relaxosome assembly involves recruitment of the catalytic relaxase to its DNA strand-specific nicking site within oriT. Here, we present evidence by AlphaFold modeling, affinity pulldowns, and in vivo site-directed photocrosslinking that the TraK Ribbon–Helix–Helix DNA-binding protein recruits TraI to oriT through a dynamic interaction in which TraI's C-terminal unstructured domain (TraICTD) wraps around TraK's C-proximal tetramerization domain. Upon relaxosome assembly, conformational changes disrupt this contact, and TraICTD instead self-associates as a prerequisite for relaxase catalytic functions or substrate engagement with the transfer channel. These findings delineate key early-stage processing reactions required for conjugative dissemination of a model MGE.

Mechanosensitive ion channels play an essential role in reacting to environmental signals and sustaining cell integrity by facilitating ion flux across membranes. For obligate intracellular pathogens like microsporidia, adapting to changes in the host environment is crucial for survival and propagation. Despite representing a eukaryote of extreme genome reduction, microsporidia have expanded the gene family of mechanosensitive ion channels of small conductance (mscS) through repeated gene duplication and horizontal gene transfer. All microsporidian genomes characterized to date contain mscS genes of both eukaryotic and bacterial origin. Here, we investigated the cryo-electron microscopy structure of the bacterially derived mechanosensitive ion channel of small conductance 2 (MscS2) from Nematocida displodere, an intracellular pathogen of Caenorhabditis elegans. MscS2 is the most compact MscS-like channel known and assembles into a unique superstructure in vitro with six heptameric MscS2 channels. Individual MscS2 channels are oriented in a heterogeneous manner to one another, resembling an asymmetric, flexible six-way cross joint. Finally, we show that microsporidian MscS2 still forms a heptameric membrane channel, however the extreme compaction suggests a potential new function of this MscS-like protein.

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.

Sexual reproduction in malaria parasites is essential for their transmission to mosquitoes and offers a divergent eukaryote model to understand the evolution of sex. Through a panel of genetic screens in Plasmodium berghei, we identify 348 sex and transmission-related genes and define roles for unstudied genes as putative targets for transmission-blocking interventions. The functional data provide a deeper understanding of female metabolic reprogramming, meiosis, and the axoneme. We identify a complex of a SUN domain protein (SUN1) and a putative allantoicase (ALLC1) that is essential for male fertility by linking the microtubule organizing center to the nuclear envelope and enabling mitotic spindle formation during male gametogenesis. Both proteins have orthologs in mouse testis, and the data raise the possibility of an ancient role for atypical SUN domain proteins in coupling the nucleus and axoneme. Altogether, our data provide an unbiased picture of the molecular processes that underpin malaria parasite transmission. A record of this paper's transparent peer review process is included in the supplemental information.

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.