Biology subjects

Whitfield, G. B.

Publications and source records attributed to Whitfield, G. B..

12 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

In situ Architecture of the Tad Pilus Machine in Caulobacter crescentus

The Tight adherence (Tad) pilus is a broadly distributed and evolutionarily distinct subclass of type IV pili that mediates cell adhesion, biofilm formation, predation, and surface sensing in many bacteria, including Caulobacter crescentus, Myxococcus xanthus, Vibrio vulnificus, and Bifidobacterium breve. Tad pili undergo cycles of extension and retraction powered by a cell-envelope-embedded nanomachine. Despite their biological importance, the architecture and assembly mechanism of the Tad pilus system have remained poorly understood. Although cryo-electron tomography (cryo-ET) has elucidated the in situ structures of other type IV pilus systems, no intact Tad machine structure has previously been reported. Here, we use cryo-ET and subtomogram averaging to resolve the near-native architecture of the C. crescentus Tad pilus within the bacterial cell envelope. 3D classification further reveals multiple assembly intermediates, and integrative modelling incorporating AlphaFold3 predictions help define the spatial arrangement of all core components. The resulting structural framework gives insight into the stepwise assembly process of the C. crescentus Tad pilus machine. Altogether, our results provide an in situ architectural model of the Tad pilus machine, establishing a foundation for understanding homologous systems across a broad range of bacteria. ImportanceInvestigating the Tad pilus nanomachine in a genetically tractable, non-pathogenic organism like Caulobacter crescentus provides a powerful model for elucidating the architecture and functional dynamics of this widespread system. Insights gained from studying the Tad machinery can improve our understanding of related Tad pilus systems in pathogenic bacteria such as Aggregatibacter actinomycetemcomitans, where Tad pili are a key determinant of biofilm formation and chronic infection. Additionally, the remarkable functional diversity of Tad systems, ranging from surface sensing in C. crescentus to bacterial predation in M. xanthus, highlights their broad biological relevance. By revealing the in situ structure and assembly mechanism of the Tad pilus biosynthetic machinery, this study advances our understanding of a major class of bacterial nanomachines and may thus provide structural insights that could inform the development of new therapeutic strategies targeting pilus-mediated virulence.

microbiology↗

bioRxiv2025

Stimulation of the Caulobacter crescentus surface sensing pathway by deletion of a specialized minor pilin-like gene

Bacteria colonize surfaces through complex mechanisms of surface sensing. Pili are dynamic bacterial appendages that play an important role in this process. In Caulobacter crescentus, tension on retracting, surface-bound pili triggers the rapid synthesis of the adhesive holdfast, which permanently attaches cells to surfaces. However, the detailed mechanisms of pilus-mediated surface sensing are unclear. In this study, we used a genetic screen to isolate mutants with altered pilus activity to identify genes that may be involved in pilus-mediated surface-sensing. This screen identified cpaL, whose deletion led to reduced piliation levels, and surprisingly, a threefold increase in surface adhesion due to increased holdfast production. To understand this finding, we compared holdfast synthesis in wild-type and cpaL mutant cells under conditions that block pilus retraction. While this treatment increased holdfast production in wild-type cells by triggering the surface-sensing pathway, no increase was observed in the cpaL mutant, suggesting that mutation of cpaL maximally stimulates surface-sensing. Furthermore, when the cpaL mutant was grown in a medium that blocks the surface sensing pathway, cells exhibited decreased surface attachment and holdfast production, consistent with a role for CpaL in pilus-dependent surface sensing in C. crescentus. To better understand the function of CpaL, we analyzed its predicted structure, which suggested that CpaL is a minor pilin fused to a mechanosensitive von Willebrand factor type A (vWA) domain that could be accommodated at the pilus tip. These results collectively position CpaL as a strong candidate for a mechanosensory element in pilus-mediated surface sensing. ImportanceSurface sensing is a crucial mechanism that allows bacteria to change their behaviors to adapt to life on a surface. Surface recognition by bacteria is the initial step toward surface colonization and biofilm formation. In Caulobacter crescentus, tight adherence (Tad) pili play a key role in surface recognition and adaptation. However, the mechanism of pilus-mediated surface sensing and the proteins that influence this process remain unknown. Here, we demonstrate that CpaL, a potential pilus tip mechanosensory protein, could be the major element of Tad pilus-mediated surface attachment and colonization in C. crescentus. CpaL plays an important role in the regulation of holdfast synthesis and production upon surface contact. By identifying CpaL as a key player in the process of surface recognition, our work offers valuable insights into the mechanisms of bacterial adhesion.

microbiology↗

bioRxiv2024

Generalists in a Specialist World: Vibrio-Phages with Broad Host Range

The host range of a bacteriophage--the diversity of hosts it can infect--is central to understanding phage ecology and applications. While most well-characterized phages have narrow host ranges, broad-host-range phages represent an intriguing component of marine ecosystems. The genetic and evolutionary mechanisms driving their generalism remain poorly understood. In this study, we analyzed Schizotequatroviruses and their Vibrio crassostreae hosts, collected from an oyster farm. Schizotequatroviruses exhibit broad host ranges, large genomes (~252 kbp) encoding 26 tRNAs, and conserved genomic organization interspersed with recombination hotspots. These recombination events, particularly in regions encoding receptor-binding proteins and antidefense systems, highlight their adaptability to host resistance. Notably, some lineages demonstrated receptor-switching between OmpK and LamB, showcasing their evolutionary flexibility. Despite their broad host range, Schizotequatroviruses were rare in the environment. Their scarcity could not be attributed to burst size, which was comparable to other phages in vitro, but may result from ecological constraints or fitness trade-offs, such as their preference for targeting generalist vibrios in seawater rather than the patho-phylotypes selected in oyster farms. Our findings clarify the genetic and ecological trade-offs shaping Schizotequatrovirus generalism and provide a foundation for future phage applications in aquaculture and beyond.

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

The vprAB-ompV-virK operon of Vibrio cholerae senses antimicrobial peptides and activates the expression of multiple resistance systems

Antimicrobial peptides are small cationic molecules produced by eukaryotic cells to combat infection, as well as by bacteria for niche competition. Polymyxin B (PmB), a cationic cyclic antimicrobial peptide, is used prophylactically in livestock for infection prevention and as a last-resort treatment for multidrug-resistant bacterial infections in humans. In this study, a transcriptomic analysis in Vibrio cholerae showed that expression of the uncharacterized gene ompV is stimulated in response to PmB. We found that ompV is organized in a conserved four-gene operon with the two-component system vprAB (carRS) and virK in V. cholerae, and that these genes are also upregulated in response to PmB treatment. A virK deletion mutant was more sensitive to the human cathelicidin LL-37 than the wild-type strain, while an ompV mutant was more sensitive to PmB and LL-37, suggesting that both OmpV and VirK contribute to antimicrobial resistance in V. cholerae. This increased sensitivity to antimicrobial peptides was not due to membrane destabilization or reduced sequestration by membrane vesicles as a result of ompV deletion. Instead, our transcriptomic analysis showed that the efflux pump vexAB, a known effector of PmB resistance, was also upregulated in the presence of PmB in an ompV-dependent manner. Examination of the predicted structure of OmpV revealed a lateral opening in the {beta}-barrel wall with access to an electronegative pocket in the barrel lumen that can accommodate PmB. Such an interaction could facilitate intracellular signaling through a conformational change in OmpV. This is the first evidence of a specialized operon governing multiple systems for antimicrobial resistance in V. cholerae. Author SummaryIn this study, we identified the first specialized operon controlling multiple systems of antimicrobial resistance in V. cholerae. The operon encodes the two-component system vprAB, which activates the main mechanism of polymyxin B resistance in V. cholerae, and the uncharacterized genes ompV and virK. We provide evidence that OmpV and VirK are implicated in antimicrobial resistance and show that OmpV has a membrane-accessible lateral opening into a pocket that could accommodate the antimicrobial peptide polymyxin B. We propose that OmpV acts as an outer membrane sensor that signals the presence of antimicrobial peptides to activate the expression of the operon, leading to the activation of multiple mechanisms of resistance, including modifications of the outer membrane and the multi-drug efflux system vexAB.

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

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↗

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

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↗