Synthesis of lipid-linked precursors of the bacterial cell wall is governed by a feedback control mechanism in Pseudomonas aeruginosa.

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting carrier is distributed among competing pathways has remained unclear. Here we describe the isolation of hyperactive variants of Pseudomonas aeruginosa MraY, the enzyme that forms the first lipid-linked PG precursor. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in vitro. The activated MraY variants have substitutions that map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural and molecular dynamics results suggest that this cavity is a binding site for externalized lipid II. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism that prevents the sequestration of lipid carrier in the PG biogenesis pathway.

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.

A conserved subcomplex within the bacterial cytokinetic ring activates cell wall synthesis by the FtsW-FtsI synthase.

Cell division in bacteria is mediated by a multiprotein assembly called the divisome. A major function of this machinery is the synthesis of the peptidoglycan (PG) cell wall that caps the daughter poles and prevents osmotic lysis of the newborn cells. Recent studies have implicated a complex of FtsW and FtsI (FtsWI) as the essential PG synthase within the divisome; however, how PG polymerization by this synthase is regulated and coordinated with other activities within the machinery is not well understood. Previous results have implicated a conserved subcomplex of division proteins composed of FtsQ, FtsL, and FtsB (FtsQLB) in the regulation of FtsWI, but whether these proteins act directly as positive or negative regulators of the synthase has been unclear. To address this question, we purified a five-member Pseudomonas aeruginosa division complex consisting of FtsQLB-FtsWI. The PG polymerase activity of this complex was found to be greatly stimulated relative to FtsWI alone. Purification of complexes lacking individual components indicated that FtsL and FtsB are sufficient for FtsW activation. Furthermore, support for this activity being important for the cellular function of FtsQLB was provided by the identification of two division-defective variants of FtsL that still form normal FtsQLB-FtsWI complexes but fail to activate PG synthesis. Thus, our results indicate that the conserved FtsQLB complex is a direct activator of PG polymerization by the FtsWI synthase and thereby define an essential regulatory step in the process of bacterial cell division.

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

Pel is a GalNAc-rich bacterial polysaccharide that contributes to the structure and function of Pseudomonas aeruginosa biofilms. The pelABCDEFG operon is highly conserved among diverse bacterial species, and Pel may therefore be a widespread biofilm determinant. Previous annotation of pel gene clusters has helped us identify an additional gene, pelX, that is present adjacent to pelABCDEFG in >100 different bacterial species. The pelX gene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have used Pseudomonas protegens Pf-5 as a model to elucidate PelX function as Pseudomonas aeruginosa lacks a pelX homologue in its pel gene cluster. We found that P. protegens forms Pel-dependent biofilms; however, despite expression of pelX under these conditions, biofilm formation was unaffected in a ΔpelX strain. This observation led us to identify a pelX paralogue, PFL_5533, which we designate here PgnE, that appears to be functionally redundant to pelX In line with this, a ΔpelX ΔpgnE 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 Å resolution. The structure revealed that PelX resembles UDP-GlcNAc C4-epimerases. Using 1H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Our results indicate 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.

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

In bacteria 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 as gene duplication, loss, rearrangement, and horizontal transfer. These mechanisms can result in functional diversification, increasing the potential evolution of novel biological pathways, and enabling pre-existing pathways to adapt 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-ß-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 revealed 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 evolution-based classification also distinguishes between PNAG production from 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 (Whitfield et al PLoS Pathogens, in press) that Bacillus cereus produces a Pel-dependent biofilm that is regulated by cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP).

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. 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.

Pel Polysaccharide Biosynthesis Requires an Inner Membrane Complex Comprised of PelD, PelE, PelF, and PelG.

The Pel polysaccharide is a structural component of the extracellular matrix of Pseudomonas aeruginosa biofilms. Recent analyses suggest that Pel production proceeds via a synthase-dependent polysaccharide secretion pathway, which in Gram-negative bacteria is defined by an outer membrane ß-barrel porin, a periplasmic tetratricopeptide repeat-containing scaffold protein, and an inner membrane-embedded synthase. Polymerization is catalyzed by the glycosyltransferase domain of the synthase component of these systems, which is allosterically regulated by cyclic 3',5'-dimeric GMP (c-di-GMP). However, while the outer membrane and periplasmic components of the Pel system have been characterized, the inner membrane complex required for Pel polymerization has yet to be defined. To address this, we examined over 500 pel gene clusters from diverse species of Proteobacteria This analysis identified an invariant set of four syntenic genes, three of which, pelD, pelE, and pelG, are predicted to reside within the inner membrane, while the fourth, pelF, encodes a glycosyltransferase domain. Using a combination of gene deletion analysis, subcellular fractionation, coimmunoprecipitation, and bacterial two-hybrid assays, we provide evidence for the existence of an inner membrane complex of PelD, PelE, and PelG. Furthermore, we show that this complex interacts with PelF in order to facilitate its localization to the inner membrane. Mutations that abolish c-di-GMP binding to the known receptor domain of PelD had no effect on complex formation, suggesting that c-di-GMP binding stimulates Pel production through quaternary structural rearrangements. Together, these data provide the first experimental evidence of an inner membrane complex involved in Pel polysaccharide production.IMPORTANCE The exopolysaccharide Pel plays an important role in bacterial cell-cell interactions, surface adhesion, and protection against certain antibiotics. We identified invariant pelDEFG gene clusters in over 500 diverse proteobacterial species. Using Pseudomonas aeruginosa, we demonstrate that PelD, PelE, PelF, and PelG form a complex at the inner membrane and propose that this complex represents the previously unidentified Pel polysaccharide synthase, which is responsible for Pel polymerization and transport across the cytoplasmic membrane. We show that the formation of this complex is independent of cyclic 3',5'-dimeric GMP (c-di-GMP) binding to the receptor PelD. Collectively, these data establish the widespread Pel apparatus as a member of the synthase-dependent pathway of polysaccharide biosynthetic systems and broaden the architectural diversity of already-established bacterial polysaccharide synthases.

FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein.

The peptidoglycan cell wall is essential for the survival and morphogenesis of bacteria1. For decades, it was thought that only class A penicillin-binding proteins (PBPs) and related enzymes effected peptidoglycan synthesis. Recently, it was shown that RodA-a member of the unrelated SEDS protein family-also acts as a peptidoglycan polymerase2-4. Not all bacteria require RodA for growth; however, its homologue, FtsW, is a core member of the divisome complex that appears to be universally essential for septal cell wall assembly5,6. FtsW was previously proposed to translocate the peptidoglycan precursor lipid II across the cytoplasmic membrane7,8. Here, we report that purified FtsW polymerizes lipid II into peptidoglycan, but show that its polymerase activity requires complex formation with its partner class B PBP. We further demonstrate that the polymerase activity of FtsW is required for its function in vivo. Thus, our findings establish FtsW as a peptidoglycan polymerase that works with its cognate class B PBP to produce septal peptidoglycan during cell division.

PelA and PelB proteins form a modification and secretion complex essential for Pel polysaccharide-dependent biofilm formation in Pseudomonas aeruginosa.

The pellicle (PEL) polysaccharide is synthesized by the opportunistic pathogen Pseudomonas aeruginosa and is an important biofilm constituent critical for bacterial virulence and persistence. PEL is a cationic polymer that promotes cell-cell interactions within the biofilm matrix through electrostatic interactions with extracellular DNA. Translocation of PEL across the outer membrane is proposed to occur via PelB, a membrane-embedded porin with a large periplasmic domain predicted to contain 19 tetratricopeptide repeats (TPRs). TPR-containing domains are typically involved in protein-protein interactions, and we therefore sought to determine whether PelB serves as a periplasmic scaffold that recruits other components of the PEL secretion apparatus. In this study, we show that the TPR domain of PelB interacts with PelA, an enzyme with PEL deacetylase and hydrolase activities. Structure determination of PelB TPRs 8-11 enabled us to design systematic deletions of individual TPRs and revealed that repeats 9-14, which are required for the cellular localization of PelA with PelB are also essential for PEL-dependent biofilm formation. Copurification experiments indicated that the interaction between PelA and PelB is direct and that the deacetylase activity of PelA increases and its hydrolase activity decreases when these proteins interact. Combined, our results indicate that the TPR-containing domain of PelB localizes PelA to the PEL secretion apparatus within the periplasm and that this may allow for efficient deacetylation of PEL before its export from the cell.

Oligomeric lipoprotein PelC guides Pel polysaccharide export across the outer membrane of Pseudomonas aeruginosa.

Secreted polysaccharides are important functional and structural components of bacterial biofilms. The opportunistic pathogen Pseudomonas aeruginosa produces the cationic exopolysaccharide Pel, which protects bacteria from aminoglycoside antibiotics and contributes to biofilm architecture through ionic interactions with extracellular DNA. A bioinformatics analysis of genome databases suggests that gene clusters for Pel biosynthesis are present in >125 bacterial species, yet little is known about how this biofilm exopolysaccharide is synthesized and exported from the cell. In this work, we characterize PelC, an outer membrane lipoprotein essential for Pel production. Crystal structures of PelC from Geobacter metallireducens and Paraburkholderia phytofirmans coupled with structure-guided disulfide cross-linking in P. aeruginosa suggest that PelC assembles into a 12- subunit ring-shaped oligomer. In this arrangement, an aromatic belt in proximity to its lipidation site positions the highly electronegative surface of PelC toward the periplasm. PelC is structurally similar to the Escherichia coli amyloid exporter CsgG; however, unlike CsgG, PelC does not possess membrane-spanning segments required for polymer export across the outer membrane. We show that the multidomain protein PelB with a predicted C-terminal ß-barrel porin localizes to the outer membrane, and propose that PelC functions as an electronegative funnel to guide the positively charged Pel polysaccharide toward an exit channel formed by PelB. Together, our findings provide insight into the unique molecular architecture and export mechanism of the Pel apparatus, a widespread exopolysaccharide secretion system found in environmental and pathogenic bacteria.

Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix.

Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.

Enzymatic modifications of exopolysaccharides enhance bacterial persistence.

Biofilms are surface-attached communities of bacterial cells embedded in a self-produced matrix that are found ubiquitously in nature. The biofilm matrix is composed of various extracellular polymeric substances, which confer advantages to the encapsulated bacteria by protecting them from eradication. The matrix composition varies between species and is dependent on the environmental niche that the bacteria inhabit. Exopolysaccharides (EPS) play a variety of important roles in biofilm formation in numerous bacterial species. The ability of bacteria to thrive in a broad range of environmental settings is reflected in part by the structural diversity of the EPS produced both within individual bacterial strains as well as by different species. This variability is achieved through polymerization of distinct sugar moieties into homo- or hetero-polymers, as well as post-polymerization modification of the polysaccharide. Specific enzymes that are unique to the production of each polymer can transfer or remove non-carbohydrate moieties, or in other cases, epimerize the sugar units. These modifications alter the physicochemical properties of the polymer, which in turn can affect bacterial pathogenicity, virulence, and environmental adaptability. Herein, we review the diversity of modifications that the EPS alginate, the Pel polysaccharide, Vibrio polysaccharide, cepacian, glycosaminoglycans, and poly-N-acetyl-glucosamine undergo during biosynthesis. These are EPS produced by human pathogenic bacteria for which studies have begun to unravel the effect modifications have on their physicochemical and biological properties. The biological advantages these polymer modifications confer to the bacteria that produce them will be discussed. The expanding list of identified modifications will allow future efforts to focus on linking these modifications to specific biosynthetic genes and biofilm phenotypes.

Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

Structure of the cytoplasmic region of PelD, a degenerate diguanylate cyclase receptor that regulates exopolysaccharide production in Pseudomonas aeruginosa.

High cellular concentrations of bis-(3',5')-cyclic dimeric guanosine mono-phosphate (c-di-GMP) regulate a diverse range of phenotypes in bacteria including biofilm development. The opportunistic pathogen Pseudomonas aeruginosa produces the PEL polysaccharide to form a biofilm at the air-liquid interface of standing cultures. Among the proteins required for PEL polysaccharide production, PelD has been identified as a membrane-bound c-di-GMP-specific receptor. In this work, we present the x-ray crystal structure of a soluble cytoplasmic region of PelD in its apo and c-di-GMP complexed forms. The structure of PelD reveals an N-terminal GAF domain and a C-terminal degenerate GGDEF domain, the latter of which binds dimeric c-di-GMP at an RXXD motif that normally serves as an allosteric inhibition site for active diguanylate cyclases. Using isothermal titration calorimetry, we demonstrate that PelD binds c-di-GMP with low micromolar affinity and that mutation of residues involved in binding not only decreases the affinity of this interaction but also abrogates PEL-specific phenotypes in vivo. Bioinformatics analysis of the juxtamembrane region of PelD suggests that it contains an α-helical stalk region that connects the soluble region to the transmembrane domains and that similarly to other GAF domain containing proteins, this region likely forms a coiled-coil motif that mediates dimerization. PelD with Alg44 and BcsA of the alginate and cellulose secretion systems, respectively, collectively constitute a group of c-di-GMP receptors that appear to regulate exopolysaccharide assembly at the protein level through activation of their associated glycosyl transferases.

Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa PelD.

The production of the PEL polysaccharide in Pseudomonas aeruginosa requires the binding of bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) to the cytoplasmic GGDEF domain of the inner membrane protein PelD. Here, the overexpression, purification and crystallization of a soluble construct of PelD that encompasses the GGDEF domain and a predicted GAF domain is reported. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion method. The crystals grew as flat plates, with unit-cell parameters a = 88.3, b = 114.0, c = 61.9 Å, α = ß = γ = 90.0°. The PelD crystals exhibited the symmetry of space group P2(1)2(1)2 and diffracted to a minimum d-spacing of 2.2 Å. On the basis of the Matthews coefficient (V(M) = 2.29 Å(3) Da(-1)), it was estimated that two molecules are present in the asymmetric unit.