Surface Appendages Research Articles

Spatiotemporal pattern formation in E. coli biofilms explained by a simple physical energy balance.

While the biofilm growth mode conveys notable thriving advantages to bacterial populations, the mechanisms of biofilm formation are still strongly debated. Here, we investigate the remarkable spontaneous formation of regular spatial patterns during the growth of an Escherichia coli biofilm. These patterns reported here appear with non-motile bacteria, which excludes both chemotactic origins and other motility-based ones. We demonstrate that a minimal physical model based on phase separation describes them well. To confirm the predictive capacity of our model, we tune the cell-cell and cell-surface interactions using cells expressing different surface appendages. We further explain how F pilus-bearing cells enroll their wild type kindred, poorly piliated, into their typical pattern when mixed together. This work supports the hypothesis that purely physicochemical processes, such as the interplay of cell-cell and cell-surface interactions, can drive the emergence of a highly organized spatial structure that is potentially decisive for community fate and for biological functions.

Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation.

Toxigenic Vibrio cholerae strains, including strains in serogroups O1 and O139 associated with the clinical disease cholera, are ubiquitous in aquatic reservoirs, including fresh, estuarine, and marine environments. Humans acquire cholera by consuming water and/or food contaminated with the microorganism. The genome of toxigenic V. cholerae harbors a cholera-toxin producing prophage (CT-prophage) encoding genes that promote expression of cholera toxin. The CT-prophage in V. cholerae is flanked by two satellite prophages, RS1 and TLC. Using cell surface appendages (TCP and/or MSHA pili), V. cholerae can sequentially acquire TLC, RS1, and CTX phages by transduction; the genome of each of these phages ultimately integrates into V. cholerae’s genome in a site-specific manner. Here, we showed that a non-toxigenic V. cholerae O1 biotype El Tor strain, lacking the entire RS1-CTX-TLC prophage complex (designated as RCT: R for RS1, C for CTX and T for TLC prophage, respectively), was able to acquire RCT from donor genomic DNA (gDNA) of a wild-type V. cholerae strain (E7946) via chitin-induced transformation. Moreover, we demonstrated that a chitin-induced transformant (designated as AAS111) harboring RCT was capable of producing cholera toxin. We also showed that recA, rather than xerC and xerD recombinases, promoted the acquisition of RCT from donor gDNA by the recipient non-toxigenic V. cholerae strain. Our data document the existence of an alternative pathway by which a non-toxigenic V. cholerae O1 strain can transform to a toxigenic strain by using chitin induction. As chitin is an abundant natural carbon source in aquatic reservoirs where V. cholerae is present, chitin-induced transformation may be an important driver in the emergence of new toxigenic V. cholerae strains.

Electrophoresis as a simple method to detect deleterious actions of engineered nanoparticles on living cells

Environmental contextAttractive interactions and subsequent contacts between nanoparticles and microorganisms are the first steps of a chain of events leading to adverse effects toward cells. We show that the electrophoretic response of complex mixtures of engineered nanoparticles and bacteria reflects initial nanoparticle-mediated cell surface damage. The technique is a promising option for rapid detection of deleterious actions of nanoparticles on biological cells. AbstractThe release of engineered nanoparticles (NPs) to the environment may have profound implications for the health of aquatic biota. In this study, we show that the initial stage of the action of NPs on bacteria can be detected by the measurement of the electrophoretic fingerprints of mixed NP–cell dispersions. Such electrokinetic signatures reflect a modification of the physicochemical surface properties of both cells and NPs following changes in the organisation of the cell envelope, subsequent release of intracellular material and/or excretion of biomolecules. The demonstration is based on a thorough investigation of the electrohydrodynamic features of genetically engineered Escherichia coli bacteria with distinct surface phenotypes (presence of adhesive YeeJ large proteins or F-pili proteinaceous filaments) exposed to silica NPs (radius of 65nm) functionalised by -NH2 terminal groups. At pH 7, electrostatics prevents interactions between bacteria and SiNH2 NPs, regardless of the considered concentration of NPs (range of 0–10−2gL−1). At pH 3, electrostatically-driven interactions allow intimate contacts between NPs and bacteria. In turn, significant modulation of the electrophoretic determinants of cells and NPs are generated owing to the alteration of the cell envelope and acquisition of bio-corona by NPs. Differentiated roles of the cell surface appendages in the mediation of NP impacts are evidenced by the measured dependence of the electropherograms on cell surface phenotype and NP concentration. Cell morphology and surface roughness, evaluated by atomic force microscopy (AFM) in liquid, confirm the conditions of pH and concentration of NPs where NP–cell interactions are operational. The combination of electrokinetics and AFM further pinpoints heterogeneities in the cell response at the single cell and population scales. Altogether, the results show that electrophoresis is suitable to detect the preliminary stage of events leading to the toxicity of NPs towards microorganisms.

The type IV pilus protein PilU functions as a PilT-dependent retraction ATPase.

Type IV pili are dynamic cell surface appendages found throughout the bacteria. The ability of these structures to undergo repetitive cycles of extension and retraction underpins their crucial roles in adhesion, motility and natural competence for transformation. In the best-studied systems a dedicated retraction ATPase PilT powers pilus retraction. Curiously, a second presumed retraction ATPase PilU is often encoded immediately downstream of pilT. However, despite the presence of two potential retraction ATPases, pilT deletions lead to a total loss of pilus function, raising the question of why PilU fails to take over. Here, using the DNA-uptake pilus and mannose-sensitive haemagglutinin (MSHA) pilus of Vibrio cholerae as model systems, we show that inactivated PilT variants, defective for either ATP-binding or hydrolysis, have unexpected intermediate phenotypes that are PilU-dependent. In addition to demonstrating that PilU can function as a bona fide retraction ATPase, we go on to make the surprising discovery that PilU functions exclusively in a PilT-dependent manner and identify a naturally occurring pandemic V. cholerae PilT variant that renders PilU essential for pilus function. Finally, we show that Pseudomonas aeruginosa PilU also functions as a PilT-dependent retraction ATPase, providing evidence that the functional coupling between PilT and PilU could be a widespread mechanism for optimal pilus retraction.

Curli production enhances clay-E. coli aggregation and sedimentation

Curli are amyloid fibrils that polymerize extracellularly from curlin, a protein that is secreted by many enteric bacteria and is important for biofilm formation. Presented here is a systematic study of the effects of curli on bacteria-clay interactions. The aggregation trends of curli-producing and curli-deficient bacteria with clay minerals were followed using gradient-sedimentation experiments, Lumisizer measurements, bright-field and electron microscopy. The results revealed that curli-producing bacteria auto-aggregated into high-density flocs (1.23 g/cm3), ranging in size from 10 to 50 μm, that settle spontaneously. In contrast, curli-deficient bacteria remained relatively stable in solution as individual cells (1–2 μm, 1.18 g/cm3), even at high ionic strength (350 mM). The stability of clay suspensions mixed with curli-deficient bacteria depended on clay type and ionic strength, the general trends being consistent with the classic DLVO theory. However, suspensions of curli-producing bacteria mixed with clays were highly unstable regardless of clay type and solution chemistry, suggesting extensive interactions between the clays and the bacteria-curli aggregates. SEM measurements revealed interesting differences in morphologies of the aggregates; montmorillonite particles coated the bacterial auto-aggregates whereas the kaolinite platelets were embedded within the larger curli-bacteria aggregates. These new observations regarding the densities, aggregation trends, and morphologies of bacteria-curli and bacteria-curli-clay complexes make it clear that production of surface appendages, such as curli, need to be considered when addressing the fate, activity and transport of bacteria - particularly in aquatic environments.

Two membrane-bound transcription factors regulate expression of various type-IV-pili surface structures in Sulfolobus acidocaldarius.

In Archaea and Bacteria, gene expression is tightly regulated in response to environmental stimuli. In the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius nutrient limitation induces expression of the archaellum, the archaeal motility structure. This expression is orchestrated by a complex hierarchical network of positive and negative regulators—the archaellum regulatory network (arn). The membrane-bound one-component system ArnR and its paralog ArnR1 were recently described as main activators of archaellum expression in S. acidocaldarius. They regulate gene expression of the archaellum operon by targeting the promoter of flaB, encoding the archaellum filament protein. Here we describe a strategy for the isolation and biochemical characterization of these two archaellum regulators. Both regulators are capable of forming oligomers and are phosphorylated by the Ser/Thr kinase ArnC. Apart from binding to pflaB, ArnR but not ArnR1 bound to promoter sequences of aapF and upsX, which encode components of the archaeal adhesive pilus and UV-inducible pili system, demonstrating a regulatory connection between different surface appendages of S. acidocaldarius.

Gram-Positive Type IV Pili and Competence.

Type IV pili (T4P) are remarkable bacterial surface appendages that carry out a range of functions. Various types of T4P have been identified in bacteria and archaea, making them almost universal structures in prokaryotes. T4P are best characterized in Gram-negative bacteria, in which pilus biogenesis and T4P-mediated functions have been studied for decades. Recent advances in microbial whole-genome sequencing have provided ample evidence for the existence of T4P also in many Gram-positive species. However, comparatively little is known, and T4P in Gram-positive bacteria are just beginning to be dissected. So far, they have mainly been studied in Clostridium and Streptococcus spp. and are involved in diverse cellular processes such as adhesion, motility, and horizontal gene transfer. Here we summarize the current understanding of T4P in Gram-positive species and their functions, with particular focus on the type IV competence pilus produced by the human pathogen Streptococcus pneumoniae and its role in natural transformation.

Mericarp morphology of the tribe Selineae (Apiaceae, Apioideae) and its taxonomic implications in Korea

Mericarp morphology of 24 taxa belonging to nine genera of the tribe Selineae (Family: Apiaceae) in Korea was studied by Scanning Electron Microscopy. UPGMA and NMDS analyses were performed based on 12 morphological characters. The mericarp surface characters like mericarp shape, rib number and shape, surface pattern, surface appendages and mericarp symmetry proved useful in distinguishing the genera of the tribe Selineae.

Filming flagella and pili in action.

This month’s Under the Lens discusses the utility of live-cell fluorescence labelling to record the dynamics of bacterial surface appendages.

Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae.

Natural transformation is a broadly conserved mechanism of horizontal gene transfer in bacterial species that can shape evolution and foster the spread of antibiotic resistance determinants, promote antigenic variation and lead to the acquisition of novel virulence factors. Surface appendages called competence pili promote DNA uptake during the first step of natural transformation 1 ; however, their mechanism of action has remained unclear owing to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus-labelling method, we define the mechanism for type IV competence pilus-mediated DNA uptake during natural transformation. First, we show that type IV competence pili bind to extracellular double-stranded DNA via their tip and demonstrate that this binding is critical for DNA uptake. Next, we show that type IV competence pili are dynamic structures and that pilus retraction brings tip-bound DNA to the cell surface. Finally, we show that pilus retraction is spatiotemporally coupled to DNA internalization and that sterically obstructing pilus retraction prevents DNA uptake. Together, these results indicate that type IV competence pili directly bind to DNA via their tip and mediate DNA internalization through retraction during this conserved mechanism of horizontal gene transfer.

A Tad pilus promotes the establishment and resistance of Vibrio vulnificus biofilms to mechanical clearance

Vibrio vulnificus is autochthonous to estuaries and warm coastal waters. Infection occurs via open wounds or ingestion, where its asymptomatic colonization of seafood, most infamously oysters, provides a gateway into the human food chain. Colonization begins with initial surface contact, which is often mediated by bacterial surface appendages called pili. Type IV Tad pili are widely distributed in the Vibrionaceae, but evidence for a physiological role for these structures is scant. The V. vulnificus genome codes for three distinct tad loci. Recently, a positive correlation was demonstrated between the expression of tad-3 and the phenotypes of a V. vulnificus descendent (NT) that exhibited increased biofilm formation, auto-aggregation, and oyster colonization relative to its parent. However, the mechanism by which tad pilus expression promoted these phenotypes was not determined. Here, we show that deletion of the tad pilin gene (flp) altered the near-surface motility profile of NT cells from high curvature, orbital retracing patterns characteristic of cells actively probing the surface to low curvature traces indicative of wandering and diminished bacteria–surface interactions. The NT flp pilin mutant also exhibited decreased initial surface attachment, attenuated auto-aggregation and formed fragile biofilms that disintegrated under hydrodynamic flow. Thus, the tad-3 locus, designated iam, promoted initial surface attachment, auto-aggregation and resistance to mechanical clearance of V. vulnificus biofilms. The prevalence of tad loci in the Vibrionaceae suggests that they may play equally important roles in other family members.

Versatile cell surface structures of archaea.

Archaea are ubiquitously present in nature and colonize environments with broadly varying growth conditions. Several surface appendages support their colonization of new habitats. A hallmark of archaea seems to be the high abundance of type IV pili (T4P). However, some unique non T4 filaments are present in a number of archaeal species. Archaeal surface structures can mediate different processes such as cellular surface adhesion, DNA exchange, motility and biofilm formation and represent an initial attachment site for infecting viruses. In addition to the functionally characterized archaeal T4P, archaeal genomes encode a large number of T4P components that might form yet undiscovered surface structures with novel functions. In this review, we summarize recent advancement in structural and functional characterizations of known archaeal surface structures and highlight the diverse processes in which they play a role.

Electron microscopic observations of prokaryotic surface appendages.

Prokaryotic microbes possess a variety of appendages on their cell surfaces. The most commonly known surface appendages of bacteria include flagella, pili, curli, and spinae. Although archaea have archaella (archaeal flagella) and various types of pili that resemble those in bacteria, cannulae, and hami are unique to archaea. Typically involved in cell motility, flagella, the thickest appendages, are 20-26 nm and 10-14 nm wide in bacteria and archaea, respectively. Bacterial and archaeal pili are distinguished by their thin, short, hair-like structures. Curli appear as coiled and aggregative thin fibers, whereas spinae are tubular structures 50-70 nm in diameter in bacteria. Cannulae are characterized by ∼25 nm-wide tubules that enter periplasmic spaces and connect neighboring archaeal cells. Hami are 1-3 μm in length and similar to barbed grappling hooks for attachment to bacteria. Recent advances in specimen preparation methods and image processing techniques have made cryo-transmission electron microscopy an essential tool for in situ structural analysis of microbes and their extracellular structures.

Numerical study of the effect of soft layer properties on bacterial electroporation

We present a numerical model of electroporation in a gram-positive bacterium, which accounts for the presence of a negatively charged soft polyelectrolyte layer (which may include a periplasmic space, peptidoglycan layer, cilia, flagella, and other surface appendages) surrounding its plasma membrane. We model the ion transport within and outside the soft layer using the soft layer electrokinetics-based Poisson-Nernst-Planck formalism. Additionally, we model the electroporation dynamics on the plasma membrane using the pore nucleation-based electroporation formalism developed by Krassowska and Filev. We find that ion transport within the soft layer (surface conduction), which depends on the relative importance of the soft layer charged group concentration compared to the buffer concentration, significantly alters the transmembrane voltage across the plasma membrane and hence the pore characteristics. Our numerical simulations suggest that surface conduction significantly lowers the pore number in the plasma membrane. This observation is consistent with experimental studies that show that gram-positive bacteria, in general, have lower transformation efficiencies compared to gram-negative bacteria. Our studies highlight a strong dependence of bacterial electroporation on cell envelope properties and buffer conditions, which need to be taken into consideration when designing electroporation protocols.

Predicting and Interpreting the Structure of Type IV Pilus of Electricigens by Molecular Dynamics Simulations.

Nanowires that transfer electrons to extracellular acceptors are important in organic matter degradation and nutrient cycling in the environment. Geobacter pili of the group of Type IV pilus are regarded as nanowire-like biological structures. However, determination of the structure of pili remains challenging due to the insolubility of monomers, presence of surface appendages, heterogeneity of the assembly, and low-resolution of electron microscopy techniques. Our previous study provided a method to predict structures for Type IV pili. In this work, we improved on our previous method using molecular dynamics simulations to optimize structures of Neisseria gonorrhoeae (GC), Neisseria meningitidis and Geobacter uraniireducens pilus. Comparison between the predicted structures for GC and Neisseria meningitidis pilus and their native structures revealed that proposed method could predict Type IV pilus successfully. According to the predicted structures, the structural basis for conductivity in G.uraniireducens pili was attributed to the three N-terminal aromatic amino acids. The aromatics were interspersed within the regions of charged amino acids, which may influence the configuration of the aromatic contacts and the rate of electron transfer. These results will supplement experimental research into the mechanism of long-rang electron transport along pili of electricigens.

Pilus biogenesis of Gram-positive bacteria: Roles of sortases and implications for assembly.

Successful adherence, colonization, and survival of Gram-positive bacteria require surface proteins, and multiprotein assemblies called pili. These surface appendages are attractive pharmacotherapeutic targets and understanding their assembly mechanisms is essential for identifying a new class of 'anti-infectives' that do not elicit microbial resistance. Molecular details of the Gram-negative pilus assembly are available indepth, but the Gram-positive pilus biogenesis is still an emerging field and investigations continue to reveal novel insights into this process. Pilus biogenesis in Gram-positive bacteria is a biphasic process that requires enzymes called pilus-sortases for assembly and a housekeeping sortase for covalent attachment of the assembled pilus to the peptidoglycan cell wall. Emerging structural and functional data indicate that there are at least two groups of Gram-positive pili, which require either the Class C sortase or Class B sortase in conjunction with LepA/SipA protein for major pilin polymerization. This observation suggests two distinct modes of sortase-mediated pilus biogenesis in Gram-positive bacteria. Here we review the structural and functional biology of the pilus-sortases from select streptococcal pilus systems and their role in Gram-positive pilus assembly.

Особливості епідемічної та епізоотичної ситуації щодо Pseudomonas aeruginosa

Особливості епідемічної та епізоотичної ситуації щодо Pseudomonas aeruginosa

Passive control of a falling sphere by elliptic-shaped appendages

The majority of investigations characterizing the motion of single or multiple particles in fluid flows consider canonical body shapes, such as spheres, cylinders, discs, etc. However, protrusions on bodies -- being either as surface imperfections or appendages that serve a function -- are ubiquitous in both nature and applications. In this work, we characterize how the dynamics of a sphere with an axis-symmetric wake is modified in the presence of thin three-dimensional elliptic-shaped protrusions. By investigating a wide range of three-dimensional appendages with different aspect ratios and lengths, we clearly show that the sphere with an appendage may robustly undergo an inverted-pendulum-like (IPL) instability. This means that the position of the appendage placed behind the sphere and aligned with the free-stream direction is unstable, in a similar way that an inverted pendulum is unstable under gravity. Due to this instability, non-trivial forces are generated on the body, leading to turn and drift, if the body is free to fall under gravity. Moreover, we identify the aspect ratio and length of the appendage that induces the largest side force on the sphere, and therefore also the largest drift for a freely falling body. Finally, we explain the physical mechanisms behind these observations in the context of the IPL instability, i.e., the balance between surface area of the appendage exposed to reversed flow in the wake and the surface area of the appendage exposed to fast free-stream flow.

Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis.

ABSTRACT Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa. In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication.

Motility and adhesion through type IV pili in Gram-positive bacteria.

Type IV pili are hair-like bacterial surface appendages that play a role in diverse processes such as cellular adhesion, colonization, twitching motility, biofilm formation, and horizontal gene transfer. These extracellular fibers are composed exclusively or primarily of many copies of one or more pilin proteins, tightly packed in a helix so that the highly hydrophobic amino-terminus of the pilin is buried in the pilus core. Type IV pili have been characterized extensively in Gram-negative bacteria, and recent advances in high-throughput genomic sequencing have revealed that they are also widespread in Gram-positive bacteria. Here, we review the current state of knowledge of type IV pilus systems in Gram-positive bacterial species and discuss them in the broader context of eubacterial type IV pili.