Biology subjects

Alva, V.

Publications and source records attributed to Alva, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Molecular architecture of colossal surface layers from hyperthermophilic archaea

Surface layers (S-layers) are paracrystalline protein lattices that form the outermost layer of the cell envelope in most archaea, providing structural support, protecting against external insults, and co-ordinating interactions with their environment. Despite their widespread occurrence, the molecular and structural details of S-layer architecture in hyperthermophilic archaea remain largely unknown. Here, we report the structure and cellular architecture of the S-layer from the hyperthermophilic archaeon Pyrobaculum arsenaticum by combining in situ electron cryotomography with single-particle electron cryomicroscopy, AlphaFold modelling, and peptide-fingerprinting mass spectrometry. We show that the S-layer is formed by an uncharacterised 292-kDa S-layer protein (SLP) extending 37 nm from the cytoplasmic membrane, making it, to our knowledge, the largest SLP structurally characterised to date. This SLP has a remarkable multidomain architecture comprising 19 immunoglobulin-like domains, 14 canonical and five non-canonical, organised into a lattice-forming core, a stalk, and a unique crown domain that stabilise the S-layer. Comparative genomic analyses unearthed homologous colossal SLP candidates across Thermoproteota, indicating that this distinctive architecture is conserved across diverse archaeal lineages. Together, our findings provide a structural framework for understanding the cell-surface organisation in hyperthermophilic archaea and suggest that these colossal S-layers represent a specialised adaptation to life at high temperatures.

microbiology↗

bioRxiv2026

A conserved archaeal ribosome-associated factor linking bacterial hibernation and eukaryotic energy sensing

Ribosome hibernation helps cells survive stress by reversibly silencing translation and limiting degradation of ribosomal complexes. Although well characterized in bacteria and eukaryotes, archaeal hibernation remains poorly understood. Using cryoEM to analyze lysates from a model archaeon, we identified AHA (AMPK{gamma}-HPF from Archaea), a broadly conserved ribosome-associated protein factor composed of two distinct modules. Structural analyses showed that AHAs C-terminal domain binds the small subunit, while its N-terminal region recognizes the large subunit, occluding the mRNA channel and the A- and P-tRNA binding sites and thereby enforcing translational silencing. Consistent with this proposed function, {Delta}AHA cells displayed reduced viability, depletion of ribosomal proteins during stationary phase, and impaired recovery upon return to growth. Phylogenetic analyses revealed that AHAs C-terminal domain shares homology with the bacterial Hibernation Promoting Factor (HPF), indicating an origin in the last universal common ancestor (LUCA) and thereby identifying HPF as a universal hibernation module. Strikingly, we observed two AMP molecues bound to AHAs N-terminal CBS-tetrad, which we found was structurally and evolutionary related to the eukaryotic energy sensor AMPK{gamma}, thus linking energy sensing between archaea and eukaryotes. Together, these findings uncover a widespread archaeal ribosome hibernation factor and establish a direct evolutionary link between prokaryotic translational silencing and eukaryotic energy sensing.

biochemistry↗

bioRxiv2026

PKN is a sex- and species-specific fertilization factor in brown algae

Fertilization, the union of male and female gametes, is central to sexual reproduction, yet the molecular mechanisms that ensure partner recognition and enforce species specificity remain elusive. Here we identify PKN, a previously uncharacterized transmembrane protein expressed exclusively in female gametes of brown algae, as an essential determinant of fertilization. Loss of PKN abolishes fertilization without affecting earlier mating steps, including gamete attraction, placing its function as a key mediator of male-female recognition. PKN contains extracellular {beta}-propeller and mucin-like domains that are enriched in predicted glycosylation sites and rapidly evolving, consistent with a role in species-specific cell-cell recognition. Notably, PKN enforces reproductive isolation within the genus Scytosiphon by preventing interspecific fertilization. Together, these findings uncover a female-encoded recognition mechanism in brown algae and reveal protein-glycan interfaces as a conserved strategy for enforcing sex- and species-specific fertilization across Eukaryotes.

developmental biology↗

bioRxiv2026

From fibril to framework: P. abyssi AbpX illuminates a calcium-responsive family of microbial biomatrix proteins that form thermostable hydrogels

Evolutionary pressure on microbial communities propagating under extreme environmental conditions often results in unique structural adaptations to promote cell survival. Here, we report an investigation of AbpX, a biomatrix protein identified in cultures of the hyperthermophilic archaeon Pyrodictium abyssi. Under ex vivo and in vitro conditions, AbpX assembles into a para-crystalline lattice composed of semiflexible fibrils. CryoEM analysis of recombinant AbpX fibrils reveals that the precursor protein polymerizes through donor strand complementation (DSC), a process previously reported for chaperone-usher fimbriae in Gram-negative bacteria. Unlike the latter DSC protein polymers, AbpX undergoes chaperone-free polymerization in the presence of calcium ions, which are sequestered at the donor strand-acceptor groove interface between protomers in the fibril. Using a combination of cryoEM and crystallographic structural information, an atomic model is proposed for the AbpX lattice that provides insight into its potential role in biofilm formation. These findings suggest that calcium ion coordination triggers fibril assembly and pre-organizes the fibrils for incorporation into the protein lattice. Bioinformatic analysis indicates that AbpX exemplifies a distinct and broadly distributed clade of calcium ion responsive biomatrix proteins within the TasA superfamily that can be fabricated into hydrogel biomaterials in vitro under environmentally benign conditions. SignificanceBiofilms provide a protective environment for microbes that enhances resilience against environmental stressors. Secreted protein filaments constitute a major structural component of these extracellular matrices, however limited information is available on the mechanism of biofilm formation and structure of the resultant protein assemblies. Here, we report a class of biomatrix proteins that are widely distributed in bacteria and archaea. We demonstrate that one such protein, P. abyssi AbpX, self-assembles into fibrils and subsequently into para-crystalline lattices in response to the presence of calcium ion. We describe a mechanistic model for the structural evolution of the fibrils into an ordered protein framework that mimics the lattice structure of the ex vivo assembly observed during cell culture.

biophysics↗

bioRxiv2025

Archaeal histone HTkC hypercompacts DNA

Histones are important organizers of chromatin in eukaryotes and archaea. In eukaryotes, the core histones assemble with DNA to form the octameric nucleosome. In archaea, histones form hypernucleosomes that are not restricted to an octameric histone core but can extend to variable lengths. We previously identified face-to-face (FtF) histones as a widely distributed group of archaeal histones that assemble into toroidal tetramer structures, distinct from nucleosomal histones. Here, we characterize the FtF histone HTkC from Thermococcus kodakarensis, which also encodes the canonical histones HTkA and HTkB. We show that HTkC wraps DNA around its toroidal tetramer and forms highly compact nucleoprotein complexes, achieving a level of compaction approximately twice that of hypernucleosomes. Consistent with a major chromatin-organizing role, htkC is among the most highly expressed genes in T. kodakarensis and its deletion leads to impaired growth. Together, these findings establish FtF histones as important organizers of archaeal chromatin alongside classical histones.

biochemistry↗

bioRxiv2025

Latent endogenous viral elements drive active infection and inheritance in a multicellular host

Endogenous viral elements (EVEs) inserted in host genomes are often regarded as inert relics of past infections. Whether they can retain infective potential and contribute to active viral cycles has remained unresolved. Here, we demonstrate that EVEs in the brown alga Ectocarpus can reactivate and drive productive viral infections. Using long-read sequencing and transcriptomics, we identify full-length, transcriptionally active giant viruses integrated within the host genome. Reactivation of these elements is specific to reproductive cells, transforming gametangia into virus-producing structures, and viral symptoms strictly correlate to the presence of active EVEs. Genetic analyses show that these elements are stably inherited, while their activation is precisely regulated by developmental and environmental cues. By resolving the genomic integration sites, we propose a mechanism for Phaeovirus integration and replication. This work provides the first direct evidence that giant EVEs can reactivate, replicate, and transmit both horizontally and vertically in a multicellular eukaryote, establishing a new model to explore latency, inheritance and the evolutionary impact of large dsDNA viruses.

genetics↗

bioRxiv2025

DNA Wrapping by a Tetrameric Bacterial Histone

Histones are conserved DNA-packaging proteins found across all domains of life. In eukaryotes, canonical histones form octamers that wrap [~]147 base pairs of DNA into nucleosomes -- the fundamental units of chromatin. In archaea, histones form dimers that further multimerize into extended hypernucleosomes along DNA. Although bacteria were long thought to lack histones, recent studies have uncovered histone homologs in diverse bacterial lineages, many of which possess key DNA-binding features. We previously characterized HBb, a bacterial histone from Bdellovibrio bacteriovorus, which binds DNA as a dimer and induces bending. Here, we describe HLp from Leptospira perolatii, a representative of a distinct bacterial histone group. Crystallographic and biophysical analyses reveal that HLp forms stable tetramers. Like HBb, HLp binds DNA non-specifically; however, it adopts a different mode of interaction -- wrapping [~]60 base pairs of DNA around its tetrameric core. This wrapping mode, supported by molecular dynamics simulations and DNA-binding assays, promotes DNA compaction and alters its topology. When expressed heterologously in Escherichia coli, HLp reorganizes nucleoid morphology, consistent with a role in chromatin organization. These findings expand the known repertoire of histone-DNA interaction in bacteria and underscore the structural and functional diversity of histone-based genome organization across the tree of life.

biochemistry↗

bioRxiv2024

Donor Strand Complementation and Calcium Ion Coordination Drive the Chaperone-free Polymerization of Archaeal Cannulae

Cannulae are tubular protein filaments that accumulate on the extracellular surface of the hyperthermophilic archaeon Pyrodictium abyssi during cell division. Cannulae have been postulated to act as a primitive extracellular matrix through which cells could communicate or exchange material, although their native biological function remains obscure. Here, we report cryoEM structural analyses of ex vivo cannulae and of in vitro protein assemblies derived from recombinant cannula-like proteins. Three-dimensional reconstructions of P. abyssi cannulae revealed that the structural interactions between protomers in the native and recombinant filaments were based on donor strand complementation, a form of non-covalent polymerization in which a donor {beta}-strand from one subunit is inserted into an acceptor groove in a {beta}-sheet of a neighboring subunit. Donor strand complementation in cannulae is reinforced through calcium ion coordination at the interfaces between structural subunits in the respective assemblies. While donor strand complementation occurs during the assembly of chaperone-usher pili, this process requires the participation of accessory proteins that are localized in the outer membrane. In contrast, we demonstrate that calcium ions can induce assembly of cannulae in the absence of other co-factors. Crystallographic analysis of a recombinant cannula-like protein monomer provided evidence that calcium ion binding primes the precursor for donor strand invasion through unblocking of the acceptor groove. Bioinformatic analysis suggested that structurally homologous cannula-like proteins occurred within the genomes of other hyperthermophilic archaea and were encompassed within the TasA superfamily of biomatrix proteins. CryoEM structural analyses of tubular filaments derived from in vitro assembly of a recombinant cannula-like protein from an uncultured Hyperthermus species revealed a common mode of assembly to the Pyrodictium cannulae, in which donor strand complementation and calcium ion binding stabilized longitudinal and lateral assembly in tubular 2D sheets.

microbiology↗

bioRxiv2024

ComFB, a new widespread family of c-di-NMP receptor proteins

Cyclic dimeric GMP (c-di-GMP) is a widespread bacterial second messenger that controls a variety of cellular functions, including protein and polysaccharide secretion, motility, cell division, cell development, and biofilm formation, and contributes to the virulence of some important bacterial pathogens. While the genes for diguanylate cyclases and c-di-GMP hydrolases (active or mutated) can be easily identified in microbial genomes, the list of c-di-GMP receptor domains is quite limited, and only two of them, PliZ and MshEN, are found across multiple bacterial phyla. Recently, a new c-di-GMP receptor protein, named CdgR or ComFB, has been identified in cyanobacteria and shown to regulate their cell size and, more recently, natural competence. Sequence and structural analysis indicated that CdgR is part of a widespread ComFB protein family, named after the "late competence development protein ComFB" from Bacillus subtilis. This prompted the suggestion that ComFB and ComFB-like proteins could also be c-di-GMP receptors. Indeed, we revealed that ComFB proteins from Gram-positive B. subtilis and Thermoanaerobacter brockii were able to bind c-di-GMP with high-affinity. The ability to bind c-di-GMP was also demonstrated for the ComFB proteins from clinically relevant Gram-negative bacteria Vibrio cholerae and Treponema denticola. These observations indicate that the ComFB family serves as yet another widespread family of bacterial c-di-GMP receptors. Incidentally, some ComFB proteins were also capable of c-di-AMP binding, identifying them as a unique family of c-di-NMP receptor proteins. The overexpression of comFB in B. subtilis, combined with an elevated concentration of c-di-GMP, suppressed motility, attesting to the biological relevance of ComFB as a c-di-GMP binding protein. IMPORTANCEThe cellular content of the bacterial second messenger c-di-GMP is controlled by c-di-GMP synthases (GGDEF domains) and hydrolases (EAL or HD-GYP domains), whose activities, in turn, respond to the signals perceived by their upstream sensory domains. Cyclic-di-GMP transmits the signals to a variety of its targets, which may contain inactivated GGDEF, EAL, or HD-GYP domains, widespread PilZ or MshEN domains, or various lineage-specific c-di-GMP receptors. Many organisms encode multiple GGDEF domains but few c-di-GMP-binding proteins, suggesting the existence of still unidentified c-di-GMP receptors. Here, we demonstrate that the ComFB family proteins, which include the recently characterized cyanobacterial CdgR/ComFB, constitute yet another widespread family of bacterial c-di-NMP receptors. We additionally show that ComFB controls bacillar motility in a c-di-GMP-dependent manner.

microbiology↗

bioRxiv2024

Tissue-Like Multicellular Development Triggered by Mechanical Compression in Archaea

The advent of clonal multicellularity is a critical evolutionary milestone, seen often in eukaryotes, rarely in bacteria, and only once observed in archaea. We show that uniaxial compression induces clonal multicellularity in haloarchaea, forming tissue-like structures. These archaeal tissues are mechanically and molecularly distinct from their unicellular lifestyle, mimicking several eukaryotic features. Archaeal tissues undergo a multinucleate stage followed by tubulin-independent cellularization, orchestrated by active membrane tension at a critical cell size. After cellularization, tissue junction elasticity become akin to animal tissues and give rise to two cell types--radial peribasal and central apicobasal cells-- with distinct actin and protein glycosylation polarity patterns. Our findings highlight the potential convergent evolution of a biophysical mechanism in the emergence of multicellular systems across domains of life.

cell biology↗

bioRxiv2024

Mapping the ultrastructural topology of the corynebacterial cell surface

Corynebacterium glutamicum is a diderm bacterium extensively used in the industrial-scale production of amino acids. Corynebacteria belong to the bacterial family Mycobacteriaceae, which is characterized by a highly unusual cell envelope with an outer membrane consisting of mycolic acids. Despite the occurrence of this distinctive cell envelope in several bacterial pathogens, including Corynebacterium diphtheriae, Mycobacterium tuberculosis, and Mycobacterium leprae, its ultrastructural and molecular details remain elusive. To address this, we investigated the cell envelope of C. glutamicum using electron cryotomography and cryomicroscopy of focused ion beam-milled cells. Our high-resolution images allowed us to accurately map the different components of the cell envelope into the tomographic density. Our data reveal that C. glutamicum has a variable cell envelope, with the outermost layer comprising the surface (S-)layer, which decorates the mycomembrane in a patchy manner. We further isolated and resolved the structure of the S-layer at 3.1 [A] resolution using single particle electron cryomicroscopy. Our structure shows that the S-layer of C. glutamicum is composed of a hexagonal array of the PS2 protein, which interacts directly with the mycomembrane via a coiled coil-containing anchoring segment. Bioinformatic analyses revealed that the PS2 S-layer is sparsely yet exclusively present within the Corynebacterium genus and absent in other genera of the Mycobacteriaceae family, suggesting distinct evolutionary pathways in the development of their cell envelopes. Our structural and cellular data collectively provide a high-resolution topography of the unusual C. glutamicum cell surface, features of which are shared by many pathogenic and microbiome-associated bacteria, as well as by several industrially significant bacterial species. This study, therefore, provides a strong experimental framework for understanding cell envelopes that contain mycolic acids.

microbiology↗

bioRxiv2024

Punctuated and continuous structural diversity of S-layers across the prokaryotic tree of life

Surface layers (S-layers) are two-dimensional (2D) crystalline lattices that frequently coat prokaryotic cells, playing a crucial role in protection, maintaining cellular integrity, and mediating environmental interactions. However, the molecular landscape of these abundant proteins has remained underexplored due to a lack of structural data. By employing AlphaFold2multimer together with planar symmetry constraints in a workflow validated by electron cryomicroscopy structure determination, we have elucidated the lattice structures of over 150 S-layers from diverse archaea and bacteria. Our findings unveil a multifaceted evolutionary landscape for S-layer proteins, highlighting key differences in the evolution of bacterial and archaeal S-layers. Our study allows us to discover underlying patterns in S-layer structure, organisa-tion, and cell anchoring mechanisms across the prokaryotic tree of life, deepening our understanding of the intricately complex microbial cell surfaces, which appear to have evolved proteinaceous S-layers independently on multiple occasions. This work will open avenues for rational manipulation of prokaryotic cellular interactions in multicellular microbiomes, as well as for innovative 2D biomaterial design.

molecular biology↗

bioRxiv2024

Halofilins as Emerging Bactofilin Families of Archaeal Cell Shape Plasticity Orchestrators

Bactofilins are rigid, non-polar bacterial cytoskeletal filaments that link cellular processes to specific curvatures of the cytoplasmic membrane. Although homologs of bactofilins have been identified in archaea and eukaryotes, functional studies have remained confined to bacterial systems. Here, we characterize representatives of two new families of archaeal bactofilins from the pleomorphic archaeon Haloferax volcanii, halofilin A (HalA) and halofilin B (HalB). HalA and HalB polymerize in vitro, assembling into straight bundles. HalA polymers are highly dynamic and accumulate at positive membrane curvatures in vivo, whereas HalB forms more static foci that localize in areas of local negative curvatures on the outer cell surface. Gene deletions and live-cell imaging show that halofilins are critical in maintaining morphological integrity during shape transition from disk (sessile) to rod (motile). Morphological defects in {Delta}halA result in accumulation of highly positive curvatures in rods but not in disks. Conversely, disk-shaped cells are exclusively affected by halB deletion, resulting in flatter cells. Furthermore, while {Delta}halA and {Delta}halB cells imprecisely determine the future division plane, defects arise predominantly during the disk-to-rod shape remodeling. In fact, the deletion of halA in the haloarchaeon Halobacterium salinarum, whose cells are consistently rod-shaped, impacted morphogenesis but not cell division. Increased levels of halofilins enforced drastic deformations in cells devoid of S-layer, suggesting that HalB polymers are more stable at defective S-layer lattice regions. Our results set halofilins apart from their bacterial correlate, where they provide mechanical scaffolding instead of directing envelope synthesis.

microbiology↗

bioRxiv2023

Atomic resolution structure of a DNA-binding histone protein from the bacterium Bdellovibrio bacteriovorus

Histones are essential for genome compaction and transcription regulation in eukaryotes, where they assemble into octamers to form the nucleosome core. In contrast, archaeal histones assemble into dimers that form hypernucleosomes upon DNA binding. Although histone homologs have been identified in bacteria recently, their DNA-binding characteristics remain largely unexplored. Our study reveals that the bacterial histone HBb (Bd0055) is indispensable for the survival of Bdellovibrio bacteriovorus, suggesting critical roles in DNA organization and gene regulation. By determining crystal structures of free and DNA-bound HBb, we unveil its distinctive dimeric assembly, diverging from those of eukaryotic and archaeal histones, while also elucidating how it binds and bends DNA through interaction interfaces reminiscent of eukaryotic and archaeal histones. Building on this, by employing various biophysical and biochemical approaches, we further substantiated the ability of HBb to bind and compact DNA by bending in a sequence-independent manner. Finally, using DNA affinity purification and sequencing, we reveal that HBb binds along the entire genomic DNA of B. bacteriovorus without sequence specificity. These distinct DNA-binding properties of bacterial histones, showcasing remarkable similarities yet significant differences from their archaeal and eukaryotic counterparts, highlight the diverse roles histones play in DNA organization across all domains of life. SummaryHistones, traditionally known for organizing and regulating DNA in eukaryotes and archaea, have recently been discovered in bacteria, opening up a new frontier in our understanding of genome organization across the domains of life. Our study investigates the largely unexplored DNA-binding properties of bacterial histones, focusing on HBb in Bdellovibrio bacteriovorus. We reveal that HBb is essential for bacterial survival and exhibits DNA-binding properties similar to archaeal and eukaryotic histones. However, unlike eukaryotic and archaeal histones, which wrap DNA, HBb bends DNA without sequence specificity. This work not only broadens our understanding of DNA organization across different life forms but also suggests that bacterial histones may have diverse roles in genome organization.

molecular biology↗

bioRxiv2022

pLM-BLAST - distant homology detection based on direct comparison of sequence representations from protein language models

MotivationThe detection of homology through sequence comparison is a typical first step in the study of protein function and evolution. In this work, we explore the applicability of protein language models to this task. ResultsWe introduce pLM-BLAST, a tool inspired by BLAST, that detects distant homology by comparing single-sequence representations (embeddings) derived from a protein language model, ProtT5. Our benchmarks reveal that pLM-BLAST maintains a level of accuracy on par with HHsearch for both highly similar sequences (with over 50% identity) and markedly divergent sequences (with less than 30% identity), while being significantly faster. Additionally, pLM-BLAST stands out among other embedding-based tools due to its ability to compute local alignments. We show that these local alignments, produced by pLM-BLAST, often connect highly divergent proteins, thereby highlighting its potential to uncover previously undiscovered homologous relationships and improve protein annotation. Availability and ImplementationpLM-BLAST is accessible via the MPI Bioinformatics Toolkit as a web server for searching precomputed databases (https://toolkit.tuebingen.mpg.de/tools/plmblast). It is also available as a standalone tool for building custom databases and performing batch searches (https://github.com/labstructbioinf/pLM-BLAST).

bioinformatics↗

bioRxiv2022

Interdigitated immunoglobulin arrays form the hyperstable surface layer of the extremophilic bacterium Deinococcus radiodurans

Deinococcus radiodurans is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, partly because of its complex cell envelope encapsulated within a hyperstable surface layer (S-layer). Despite decades of research into this cell envelope, atomic structural details of the S-layer have remained obscure. In this study, we report the electron cryomicroscopy structure of the D. radiodurans S-layer, showing how it is formed by the Hexagonally Packed Intermediate-layer (HPI) protein arranged in a planar hexagonal lattice. The HPI protein forms an array of immunoglobulin-like folds within the S-layer, with each monomer extending into the adjoining hexamer, leading to a highly interconnected, stable, sheet-like arrangement. Using electron cryotomography and subtomogram averaging from focused ion beam-milled D. radiodurans cells, we obtained a structure of the cellular S-layer, showing how this HPI S-layer coats native membranes on the surface of cells. Our S-layer structure from the diderm bacterium D. radiodurans shows similarities to immunoglobulin-like domain-containing S-layers from monoderm bacteria and archaea, highlighting shared traits in cell surface organization across different domains of life, with connotations on the evolution of immunoglobulin-based molecular recognition systems in eukaryotes.

microbiology↗

bioRxiv2022

A multi-domain connector links the outer membrane and cell wall in deep-branching bacteria

Deinococcus radiodurans is a deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of D. radiodurans has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in D. radiodurans and related bacteria. Here, we report an electron cryomicroscopy (cryo-EM) structure of the abundant D. radiodurans OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded {beta}-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Using the power of structure prediction and sequence-based bioinformatics, we further show that SlpA-like proteins are widespread in deep-branching Gram-negative bacteria, plausibly constituting an ancestral superfamily of OM-PG connectors, important for organizing the cell envelopes of many bacteria. Finally, combining our atomic structures with tomography of cell envelopes, we report a model for the cell surface of D. radiodurans, with implications on understanding the cell surface organization and hyperstability of D. radiodurans and related bacteria. Furthermore, the widespread occurrence of SlpA-like OM-PG connectors in deep-branching bacteria will help in understanding the evolutionary transition between Gram-negative and Gram-positive bacteria.

microbiology↗

bioRxiv2022

Architecture of the biofilm-associated archaic CupE pilus from Pseudomonas aeruginosa

Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogen Pseudomonas aeruginosa. We show that CupE pili consist of CupE1 subunits arranged in a zigzag architecture, with an N-terminal donor {beta}-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, resulting in an overall flexible pilus arrangement. Imaging CupE pili on the surface of P. aeruginosa cells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which may facilitate their role in promoting cohesion between bacterial cells. Finally, bioinformatic analysis shows the widespread abundance of cupE genes in isolates of P. aeruginosa and the co-occurrence of cupE with other cup clusters, suggesting interdependence of cup pili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili and their role in promoting cellular adhesion and biofilm formation in P. aeruginosa.

microbiology↗