Biology subjects

Marmont, L. S.

Publications and source records attributed to Marmont, L. S..

6 recordsLinked to original sources

bioRxiv2024

The structure of the Tad pilus alignment complex reveals a periplasmic conduit for pilus extension

The Tad (Tight adherence) pilus is a bacterial appendage implicated in bacterial virulence, cell-cell aggregation, and biofilm formation. Despite its homology to the well-characterised Type IV pilus, the structure and assembly mechanism of the Tad pilus are poorly understood. Here, we investigate the role of the protein RcpC from Pseudomonas aeruginosa. Our analyses reveal that RcpC forms a dodecameric periplasmic complex, anchored to the inner membrane by a transmembrane helix, and interacting with the outer membrane secretin RcpA. We use single-particle Cryo-EM to elucidate the structure of this RcpC dodecamer, and cell-based assays to demonstrate that the RcpC-RcpA complex is essential for Tad-mediated cell-cell aggregation. Collectively, these data demonstrate that RcpC forms the Tad pilus alignment complex, which provides a conduit across the periplasm for the Tad pilus filament to access the extracellular milieu. Our experimental data and structure-based models allow us to propose a mechanism for Tad plus assembly.

microbiology↗

bioRxiv2023

A feedback control mechanism governs the synthesis of lipid-linked precursors of the bacterial cell wall

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall, O-antigens, and capsules are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting lipid carrier is effectively distributed among competing pathways has remained unclear for some time. Here, we describe the isolation and characterization of hyperactive variants of Pseudomonas aeruginosa MraY, the essential and conserved enzyme catalyzing the formation of the first lipid-linked PG precursor called lipid I. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in a purified system. Amino acid substitutions within the activated MraY variants unexpectedly map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural evidence and molecular dynamics simulations suggest that the cavity is a binding site for lipid II molecules that have been transported to the outer leaflet of the membrane. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism to prevent the sequestration of lipid carrier in the PG biogenesis pathway. MraY belongs to the broadly distributed polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase (PNPT) superfamily of enzymes. We therefore propose that similar feedback mechanisms may be widely employed to coordinate precursor supply with demand by polymerases, thereby optimizing the partitioning of lipid carriers between competing glycan biogenesis pathways.

microbiology↗

bioRxiv2020

PelX is a UDP-N-acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation

Pel is an N-acetylgalactosamine rich polysaccharide that contributes to the structure and function of Pseudomonas aeruginosa biofilms. The pelABCDEFG operon is highly conserved among diverse bacterial species, and thus Pel may be a widespread biofilm determinant. Previous annotation of pel gene clusters led us to identify an additional gene, pelX, that is found adjacent to pelABCDEFG in over 100 different bacterial species. The pelX gene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily of enzymes, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have used Pseudomonas protegens Pf-5 as a model to understand PelX function as P. aeruginosa lacks a pelX homologue in its pel gene cluster. We find that P. protegens forms Pel-dependent biofilms, however, despite expression of pelX under these conditions, biofilm formation was unaffected in a {Delta}pelX strain. This observation led to our identification of the pelX paralogue, PFL_5533, which we designate pgnE, that appears to be functionally redundant to pelX. In line with this, a {Delta}pelX {Delta}pgnE double mutant was substantially impaired in its ability to form Pel-dependent biofilms. To understand the molecular basis for this observation, we determined the structure of PelX to 2.1[A] resolution. The structure revealed that PelX resembles UDP-N-acetylglucosamine (UDP-GlcNAc) C4-epimerases and, using 1H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Taken together, our results demonstrate that Pel-dependent biofilm formation requires a UDP-GlcNAc C4-epimerase that generates the UDP-GalNAc precursors required by the Pel synthase machinery for polymer production.

microbiology↗

bioRxiv2019

Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems (https://doi.org/10.1101/769745). Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3,5-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of Bacilli, Clostridia, Streptococci, and Actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.\n\nAuthor summaryThe Pel polysaccharide is required for biofilm formation in P. aeruginosa and we have previously found that the genes necessary for biosynthesis of this polymer are broadly distributed across Gram-negative bacteria. Herein, we show that many species of Gram-positive bacteria also possess Pel biosynthetic genes and demonstrate that these genes are used Bacillus cereus for biofilm formation. We show that Pel production in B. cereus is regulated by c-di-GMP and have identified two enzymes, a diguanylate cyclase, CdgF, and a phosphodiesterase, CdgE, that control the levels of this bacterial signalling molecule. While Pel production in B. cereus also requires the binding of c-di-GMP to the receptor PelD, the divergence of this protein in Streptococci suggests a c-di-GMP independent mechanism of regulation is used in this species. The discovery of a Pel biosynthetic gene cluster in Gram-positive bacteria and our characterization of the pel operon in B. cereus suggests that Pel is a widespread biofilm component across all bacteria.

microbiology↗

bioRxiv2019

A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries

In bacterial functionally related genes comprising metabolic pathways and protein complexes are frequently encoded in operons and are widely conserved across phylogenetically diverse species. The evolution of these operon-encoded processes is affected by diverse mechanisms such gene duplication, loss, rearrangement, and horizontal transfer. These mechanisms can result in functional diversification of gene-families, increasing the potential evolution of novel biological pathways, and serves to adapt pre-existing pathways to the requirements of particular environments. Despite the fundamental importance that these mechanisms play in bacterial environmental adaptation, a systematic approach for studying the evolution of operon organization is lacking. Herein, we present a novel method to study the evolution of operons based on phylogenetic clustering of operon-encoded protein families and genomic-proximity network visualizations of operon architectures. We applied this approach to study the evolution of the synthase dependent exopolysaccharide (EPS) biosynthetic systems: cellulose, acetylated-cellulose, poly-{beta}-1,6-N-acetyl-D-glucosamine (PNAG), Pel, and alginate. These polymers have important roles in biofilm formation, antibiotic tolerance, and as virulence factors in opportunistic pathogens. Our approach reveals the complex evolutionary landscape of EPS machineries, and enabled operons to be classified into evolutionarily distinct lineages. Cellulose operons show phyla-specific operon lineages resulting from gene loss, rearrangement, and the acquisition of accessory loci, and the occurrence of whole-operon duplications arising through horizonal gene transfer. Our evolutionary-based classification also distinguishes between the evolution of PNAG production between Gram-negative and Gram-positive bacteria on the basis of structural and functional evolution of the acetylation modification domains shared by PgaB and IcaB loci, respectively. We also predict several pel-like operon lineages in Gram-positive bacteria, and demonstrate in our companion paper (BIORXIV/2019/768473) that Bacillus cereus produces a Pel-dependent biofilm that is regulated by cyclic-3,5-dimeric guanosine monophosphate (c-di-GMP).\n\nAUTHOR SUMMARYIn bacterial genomes biological processes are frequently encoded as operons of co-transcribed neighbouring genes belonging to diverse protein families. Therefore, studying the evolution of bacterial operons provides valuable insight into understanding the biological roles of genes involved in environmental adaptation. However, no systematic approach has yet been devised to examine both the evolutionary relationships of operon encoded genes and the evolution of operon organization as a whole. To address this challenge, we present a novel method to study operon evolution by integrating phylogenetic tree based clustering and genomic-context networks. We apply this approach to perform the first systematic survey of all known synthase dependent bacterial biofilm machineries, demonstrating the generalizability of our approach for operons of diverse size, protein family composition, and species distribution. Our approach is able to identify distinct biofilm operon clades across phylogenetically diverse bacteria, resulting from gene rearrangement, duplication, loss, fusion, and horizontal gene transfer. We also elucidate different evolutionary trajectories of Gram-negative and Gram-positive biofilm production machineries, and in a companion paper (BIORXIV/2019/768473) present the experimental validation of a novel mode of biofilm production in a subset of Gram-positive bacteria.

bioinformatics↗

bioRxiv2018

FtsW is a peptidoglycan polymerase that is activated by its cognate penicillin-binding protein

The peptidoglycan cell wall is essential for the survival and shape maintenance ofbacteria.1 For decades it was thought that only penicillin-binding proteins (PBPs) effected peptidoglycan synthesis. Recently, it was shown that RodA, a member of the Rod complex involved in side wall peptidoglycan synthesis, acts as a peptidoglycan polymerase.2-4 RodA is absent or dispensable in many bacteria that contain a cell wall; however, all of these bacteria have a RodA homologue, FtsW, which is a core member of the divisome complex that is essential for septal cell wall assembly.5,6 FtsW was previously proposed flip the peptidoglycan precursor Lipid II to the peripasm,7,8 but we report here that FtsW polymerizes Lipid II. We show that FtsW polymerase activity depends on the presence of the class B PBP (bPBP) that it recruits to the septum. We also demonstrate that the polymerase activity of FtsW is required for its function in vivo. Our findings establish FtsW as a peptidoglycan polymerase that works with its cognate bPBP to produce septal peptidoglycan during cell division.

microbiology↗