Biology subjects

Howell, P. L.

Publications and source records attributed to Howell, P. L..

At least 19 recordsLinked to original sources

bioRxiv2026

Reversible biological motion with unidirectional catalysis through inversion of ATPase orientation

Biological and engineered machines generally achieve reversibility through regulated switches in the directionality of fixed-orientation motors. Bacterial Tad pilus nanomachines extend and retract pili using a single motor with a unidirectional catalytic mechanism, a capability with no precedent. Using AlphaFold3 modeling, comparative structural analyses, and pilus activity assays, we find that the Tad motor ATPase CpaF achieves bidirectionality through physical inversion; alternating which face of the ATPase toroid engages the platform complex. The two orientations contact the pilus machinery in mutually exclusive extension- or retraction-specific configurations that drive reversible pilus dynamics using the same unidirectional catalytic cycle. Retraction requires conserved C-terminal residues in CpaF whose nucleotide-driven motions oppose those of the extension interface. Thus, nature has adopted a solution for reversible movement not yet conceived by human engineering.

microbiology↗

bioRxiv2025

FimX regulates type IV pilus localization via the Pil-Chp chemosensory system in Acinetobacter baylyi

Type IV pili (T4P) are widespread dynamic appendages required for diverse prokaryotic behaviors including twitching motility, biofilm formation, and DNA uptake leading to natural transformation. Although the components involved in T4P assembly and dynamics are largely conserved across divergent clades of bacteria, the mechanisms underlying T4P function and regulation differ significantly and remain poorly characterized outside of a select few model organisms. One understudied characteristic of T4P includes the spatial organization of T4P envelope-spanning nanomachines and how organizational patterns contribute to single cell behaviors. The bacterial species Acinetobacter baylyi localizes its T4P nanomachines in a unique pattern along the long axis of the cell, making it a robust model to study the mechanisms underlying the regulation of intracellular organization in single cell organisms. In this work, we find that the T4P regulatory protein FimX has been co-opted away from regulating T4P dynamics to instead control T4P positioning through a chemosensory Pil-Chp pathway. We show FimX directly interacts with the Pil-Chp histidine kinase ChpA which likely influences Pil-Chp signaling and the subsequent positioning of T4P machines. These data contribute to our understanding of how bacterial regulatory systems can evolve to function in diverse biological processes. SIGNIFICANCE STATEMENTSubcellular organization of protein complexes plays a vital role in their function in all domains of life, yet the molecular mechanisms underlying subcellular organization in bacteria remain poorly understood. This work shows the T4P regulatory component FimX directly interacts with a chemosensory signaling protein to regulate the positioning of T4P nanomachines. These data provide insight into how signaling systems can evolve to regulate diverse processes, and they establish a direct connection between environmental signaling cascades and the regulation of bacterial cell biology.

microbiology↗

bioRxiv2024

Coordinated conformational changes in the Tad pilus ATPase CpaF facilitate a rotary mechanism of catalysis

The type IV pilus family uses PilT-like ATPases to rapidly assemble and disassemble pilin subunits. Among them, the tight adherence (Tad) pilus performs both functions using a single bifunctional ATPase, CpaF. How ATP catalysis by CpaF facilitates both assembly and disassembly of the Tad pilus remains unclear. Here, we determined electron cryo-microscopy structures of CpaF in three distinct conformations and nucleotide occupancies. Packing unit analyses revealed differences in nucleotide coordination in the active sites, as well as synchronized domain movements during the catalytic cycle. Alphafold3 modeling demonstrated that CpaF and other motors within the type IV filament superfamily use a similar binding interface to engage their respective platform proteins. From these data, we propose that CpaF employs a clockwise, rotary mechanism of catalysis, which couples chemical energy to mechanical force during the biogenesis of Tad pili, a process broadly applicable to other single motor systems.

microbiology↗

bioRxiv2024

Structural and functional analysis of Pseudomonas aeruginosa PelA provides insight into the modification of the Pel exopolysaccharide

A major biofilm matrix determinant of Pseudomonas aeruginosa is the partially deacetylated -1,4 linked N-acetylgalactosamine polymer, Pel. After synthesis and transport of the GalNAc polysaccharide across the inner membrane, PelA partially deacetylates and hydrolyzes Pel before its export out of the cell via PelB. While the Pel modification and export proteins are known to interact in the periplasm, it is unclear how the interaction of PelA and PelB coordinates these processes. To determine how PelA modifies the polymer, we determined its structure to 2.1 [A] and found a unique arrangement of four distinct domains. We have shown previously that the hydrolase domain exhibits endo--1,4-N-acetylgalactosaminidase activity. Characterization of the deacetylase domain revealed that PelA is the founding member of a new carbohydrate esterase family, CE#. Further, we found that the PelAB interaction enhances the deacetylation of N-acetylgalactosamine oligosaccharides. Using the PelA structure in conjunction with AlphaFold2 modelling of the PelAB complex, we propose a model wherein PelB guides Pel to the deacetylase domain of PelA and subsequently to the porin domain of PelB for export. Perturbation or loss of the PelAB interaction would result in less efficient deacetylation and potentially result in increased Pel hydrolysis. In PelA homologues across many phyla, the predicted structure and active sites are conserved, suggesting that there is a common modification mechanism in Gram-negative bacterial species that contain a functional pel operon.

microbiology↗

bioRxiv2024

Autoaggregation in Streptococcus intermedius is driven by the Pel polysaccharide

The Streptococcus Milleri Group (SMG) comprising of Streptococcus intermedius, anginosus and constellatus are commensal bacteria commonly found in healthy individuals. These bacteria are increasingly being recognized as opportunistic pathogens that can cause purulent infections at sterile body sites and have also been identified in the sputum of individuals with cystic fibrosis. Although the mechanisms of conversion to opportunistic pathogens are not well understood, auto-aggregation is a key driver of biofilm adhesion and cohesion in many Streptococci and Staphylococci. Here, we identify a gene cluster in the S. intermedius genome with significant homology to the pel operons in Bacillus cereus and Pseudomonas aeruginosa, which are required for Pel exopolysaccharide production and biofilm formation in these species. Characterization of a panel of clinical S. intermedius strains identified a range of aggregating phenotypes. Analysis of the pel operon in the hyper-aggregating C1365 strain revealed that each of the canonical pelDEADAFG genes, but not the four additional genes are required for aggregation. Further, we demonstrate that C1365 produces a GalNAc-rich exopolysaccharide and that aggregates can be disrupted by the 1,4 N-acetylgalactosaminidases, PelA and Sph3, but not other glycoside hydrolases, proteinase K or DNase I. Using an abscess model of mouse infection, we show that Pel driven aggregation leads to longer lasting infections, and that lack of Pel allows for the bacteria to be cleared more effectively. The polymer also affects how the bacteria interacts with the host immune system. Collectively, our data suggest that the pel operon has relevancy to S. intermedius pathogenicity.

microbiology↗

bioRxiv2023

CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and A. fumigatus mutants with altered cell wall composition, we herein found that CD56 interacts with the A. fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A. fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells.

microbiology↗

bioRxiv2023

The Metabolic Usage and Glycan Destinations of GlcNAz in E. coli

Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars such as N-acetylglucosamine (GlcNAc) being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAc-containing glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway, and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc, N-azidoacetylglucosamine (GlcNAz), for metabolic labelling in E. coli. The cytosolic production of UDP-GlcNAz was confirmed using fluorescence assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated, was interrogated by analyzing potential sites including: peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-{beta}-1,6-N-acetylglucosamine (PNAG), lipopolysaccharide (LPS) and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) towards UDP-GlcNAz in vitro and lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo confirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/553294v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1c4a9c9org.highwire.dtl.DTLVardef@66f71corg.highwire.dtl.DTLVardef@1e981bcorg.highwire.dtl.DTLVardef@14489b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well-known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro, and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.

microbiology↗

bioRxiv2023

PNAG exopolysaccharide eradication gives neutrophils access to Staphylococcus aureus biofilm infections

Staphylococcus aureus (S. aureus) can form biofilms on biotic or abiotic surfaces making biofilm infections a relevant clinical problem. Biofilms can evade immunity and resist antimicrobial treatment, and as such an understanding of biofilm infection in vivo is necessary to inform new therapeutics. Using a mouse model of S. aureus foreign-body skin infection and intravital microscopy, we imaged the interactions between neutrophils and S. aureus biofilm. We observed that neutrophils were separated from bacteria by a biofilm matrix composed of the polysaccharide intercellular adhesin (PIA), an exopolysaccharide chemically designated as poly-N-acetylglucosamine (PNAG) that is produced by enzymatic machinery encoded by the icaADBC operon. Infection with icaADBC-deficient S. aureus strains led to increased neutrophil infiltration and access to bacteria and resulted in full clearance of infection by 7 days. Moreover, enzymatic treatment with PgaB, which hydrolyzes partially deacetylated PNAG, was shown to disaggregate the biofilm giving neutrophils access into the infection site to improve clearance. Taken together, our results show that PNAG shelters S. aureus biofilms from innate host defense, and that targeting the biofilm matrix with glycoside hydrolases is a promising therapeutic avenue to treat S. aureus biofilm infections. Author SummaryStaphylococcus aureus is a major cause of biofilm-associated infections, which pose a major threat to human health. A biofilm is difficult to treat since bacteria are protected from antimicrobials within an extracellular matrix. This study is the first to show that the PgaB enzyme, a glycoside hydrolase, can disrupt the S. aureus biofilm matrix in vivo. Disrupting the biofilm matrix with PgaB gives neutrophils access to bacteria for elimination.

immunology↗

bioRxiv2022

Distinct types of multicellular aggregates in Pseudomonas aeruginosa liquid cultures

Pseudomonas aeruginosa forms suspended multicellular aggregates when cultured in liquid media. Such aggregates may be important in disease, and/or as a pathway to biofilm formation. The polysaccharide Psl and extracellular DNA (eDNA) have both been implicated in aggregation, but previous results depend strongly on the experimental conditions. Here we develop a quantitative microscopy-based method for assessing changes in the size distribution of suspended aggregates over time in growing cultures. For exponentially growing cultures of P. aeruginosa PAO1, we find that aggregation is mediated by cell-associated Psl, rather than by either eDNA or se-creted Psl. These aggregates arise de novo within the culture via a growth process that involves both collisions and clonal growth. They are "non-cheatable" since Psl non-producing cells do not aggregate with producers. In contrast, we find that stationary phase (overnight) cultures contain a different type of multicelullar aggregate, in which both eDNA and Psl mediate cohesion. Our findings suggest that the physical and bi-ological properties of multicellular aggregates may be very different in early-stage vs late-stage bacterial cultures.

microbiology↗

bioRxiv2022

Bps polysaccharide of Bordetella pertussis resists antimicrobial peptides by functioning as a dual surface shield and decoy and converts Escherichia coli into a respiratory pathogen.

Infections and disease caused by the obligate human pathogen Bordetella pertussis (Bp) are increasing, despite widespread vaccinations. The current acellular pertussis vaccines remain ineffective against nasopharyngeal colonization, carriage, and transmission. In this work, we tested the hypothesis that Bordetella polysaccharide (Bps), a member of the poly-{beta}-1,6-A-acetyl-D-glucosamine (PNAG/PGA) family of polysaccharides promotes respiratory tract colonization of Bp by resisting killing by antimicrobial peptides (AMPs). Genetic deletion of the bpsA-D locus, as well as treatment with the specific glycoside hydrolase Dispersin B, increased susceptibility to AMP-mediated killing. Bps was found to be both cell surface-associated and secreted during laboratory growth and mouse infections. Addition of bacterial supernatants containing Bps and purified Bps increased B. pertussis resistance to AMPs. By utilizing ELISA, immunoblot and flow cytometry assays, we show that Bps functions as a dual surface shield and decoy by inhibiting AMP binding. Co-inoculation of C57BL/6J mice with a Bps-proficient strain enhanced respiratory tract survival of the Bps-deficient strain. In combination, the presented results highlight the critical role of Bps as a central driver of B. pertussis pathogenesis. Heterologous production of Bps in a non-pathogenic E. coli K12 strain increased AMP resistance in vitro, and augmented bacterial survival and pathology in the mouse respiratory tract. Therefore, by conferring virulence traits across bacterial genera, Bps transforms a primarily intestinal and urinary tract bacterium into a respiratory pathogen. These studies can serve as a foundation for other PNAG/PGA polysaccharides and for the development of an effective Bp vaccine that includes Bps. Author summaryPertussis or whooping cough, caused by the obligate human pathogen Bordetella pertussis (Bp), is resurging in many countries. Currently, the mechanism by which B. pertussis subverts and resists host immunity is poorly known. In this manuscript, we examined the role of the B. pertussis polysaccharide Bps in promoting resistance to antimicrobial peptides (AMPs), a critical component of host immune defense. We show that the presence of Bps on the bacterial cell surface enhanced AMP resistance. Bps was secreted both during bacterial growth and during mouse infections. We further found that Bps functioned both as a surface shield and decoy, thereby inhibiting AMP binding. Simultaneous infection of mice with Bps-proficient and Bps- deficient strains resulted in greater survival of the Bps-deficient strain in the mouse respiratory tract. Finally, production of Bps in a non-pathogenic E. coli strain increased AMP resistance in vitro, and increased bacterial survival and heightened pathology in the mouse respiratory tract. Our study provides new insights into how B. pertussis has evolved to survive in the mammalian respiratory tract. Additionally, these studies underscore the potential of a single virulence factor to convert a non-pathogenic bacterium into a respiratory tract pathogen.

microbiology↗

bioRxiv2021

Truncation of poly-N-acetylglucosamine (PNAG) polymerization with N-acetylglucosamine analogues

Bacteria require polysaccharides for structure, survival, and virulence. Despite the central role these structures play in microbiology few tools are available to manipulate their production. In E. coli the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N3, SH, NH2) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro the inhibitors cause PNAG chain termination; consistent with the mechanism of PNAG polymerization from the non-reducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

biochemistry↗

bioRxiv2020

Pseudomonas aeruginosa uses c-di-GMP phosphodiesterases RmcA and MorA to regulate biofilm maintenance

While the early stages of biofilm formation have been well characterized, less is known about the requirements for Pseudomonas aeruginosa to maintain a mature biofilm. We utilized a P. aeruginosa-phage interaction to find that rmcA and morA, two genes which encode for c-di-GMP-degrading phosphodiesterase (PDEs) enzymes, are important for the regulation of biofilm maintenance. Deletion of these genes initially results in an elevated biofilm phenotype characterized by increased production of c-di-GMP, Pel polysaccharide and biofilm biomass. In contrast to the wild-type strain, these mutants were unable to maintain the biofilm when exposed to carbon-limited conditions. The susceptibility to nutrient limitation, and subsequent loss of biofilm viability of these mutants, was phenotypically reproduced with a stringent response mutant ({Delta}relA {Delta}spoT), indicating that the {Delta}rmcA and {Delta}morA mutants may be unable to appropriately respond to nutrient limitation. Genetic and biochemical data indicate that RmcA and MorA physically interact with the Pel biosynthesis machinery, supporting a model whereby unregulated Pel biosynthesis contributes to the death of the {Delta}rmcA and {Delta}morA mutant strains in an established biofilm when nutrient-limited. These findings provide evidence that c-di-GMP-mediated regulation is required for mature biofilms of P. aeruginosa to effectively respond to changing availability of nutrients. Furthermore, the PDEs involved in biofilm maintenance are distinct from those required for establishing a biofilm, thus indicating that a wide variety of c-di-GMP metabolizing enzymes in organisms like P. aeruginosa likely allows for discrete control over the formation, maintenance or dispersion of biofilms. ImportanceRecent advances in our understanding of c-di-GMP signaling have provided key insights into the regulation of biofilms. Despite an improved understanding of how they initially form, the processes that facilitate the long-term maintenance of these multicellular communities remain opaque. We found that P. aeruginosa requires two phosphodiesterases, RmcA and MorA, to maintain a mature biofilm and that P. aeruginosa biofilms lacking these PDEs succumb to nutrient limitation and die. The biofilm maintenance deficiency observed in {Delta}rmcA and {Delta}morA mutants was also found in the stringent response defective {Delta}relA {Delta}spoT strain, suggesting that a regulatory intersection between c-di-GMP signaling, EPS biosynthesis and the nutrient limitation response is important for persistent surface growth. We uncover components of an important regulatory system needed for P. aeruginosa to persist in nutrient-poor conditions, and provide some of the first evidence that maintaining a mature biofilm is an active process.

microbiology↗

bioRxiv2020

TsaP binds PilQ and is involved in cyclic di-GMP signaling

The type IV pilus machinery is a multi-protein complex that polymerizes and depolymerizes a pilus fibre used for attachment, twitching motility, phage adsorption, natural competence, protein secretion, and surface-sensing. An outer membrane secretin pore is required for passage of the pilus fibre out of the cell. Herein, the structure of the tetradecameric secretin, PilQ, from the Pseudomonas aeruginosa type IVa pilus system was determined to 4.3 [A] and 4.4 [A] resolution in the presence and absence of C7 symmetric spokes, respectively. The heptameric spokes were found to be the two tandem C-terminal domains of TsaP. TsaP forms a belt around PilQ and while the protein is not essential for twitching motility, over-expression of TsaP triggers a signal cascade upstream of PilY1 leading to cyclic di-GMP up-regulation. These results resolve the identity of the spokes identified with Proteobacterial PilQ homologs and may reveal a new component of the surface-sensing cyclic di-GMP signal cascade. IMPACT STATEMENTThe type IV pilus is critical for bacterial virulence. The co-structure of the pilus secretin PilQ and TsaP is determined. Characterization of TsaP implicates it in surface-sensing signal transduction.

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↗

bioRxiv2019

Multiple conformations facilitate PilT function in the type IV pilus

Type IV pilus-like systems are protein complexes that polymerize a fibre of pilins. They are critical for virulence in many pathogens. Pilin polymerization and depolymerization are powered by motor PilT-like ATPases thought to possess C2 symmetry. However, most PilT-like ATPases crystallize with either C3 or C6 symmetry and the relevance of these conformations is unclear. Here we determined the X-ray structures of PilT in four unique conformations and used these structures to classify the conformation of available PilT-like ATPase structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT revealed condition-dependent preferences for C2, C3, and C6 conformations. The physiologic importance of these conformations was validated by co-evolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data we propose a comprehensive model of PilT function with broad implications for PilT-like ATPases.

microbiology↗