RESUMEN
Xanthomonas albilineans (Xal) is a gram-negative bacterial pathogen responsible for developing sugarcane leaf scald disease, which engenders significant economic losses within the sugarcane industry. In the current study, homologous recombination exchange was carried out to induce mutations within the virB/D4-like type IV secretion system (T4SS) genes of Xal. The results revealed that the virB11-deletion mutant (ΔvirB11) exhibited a loss in swimming and twitching motility. Application of transmission electron microscopy analysis further demonstrated that the ΔvirB11 failed to develop flagella formation and type IV pilus morphology and exhibited reduced swarming behaviour and virulence. However, these alterations had no discernible impact on bacterial growth. Comparative transcriptome analysis between the wild-type Xal JG43 and the deletion-mutant ΔvirB11 revealed 123 differentially expressed genes (DEGs), of which 28 and 10 DEGs were notably associated with flagellar assembly and chemotaxis, respectively. In light of these findings, we postulate that virB11 plays an indispensable role in regulating the processes related to motility and chemotaxis in Xal.
Asunto(s)
Proteínas Bacterianas , Fimbrias Bacterianas , Flagelos , Xanthomonas , Xanthomonas/patogenicidad , Xanthomonas/genética , Virulencia/genética , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Fimbrias Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Regulación Bacteriana de la Expresión Génica , Morfogénesis , Enfermedades de las Plantas/microbiología , Saccharum/microbiologíaRESUMEN
Within the highly diverse type four filament (TFF or T4F) superfamily, the machineries of type IVa pili (T4aP) and the type 2 secretion system (T2SS) in diderm bacteria exhibit a substantial sequence similarity despite divergent functions and distinct appearances: T4aP can extend micrometers beyond the outer membrane, whereas the endopili in the T2SS are restricted to the periplasm. The determination of the structure of individual components and entire filaments is crucial to understand how their structure enables them to serve different functions. However, the dynamics of these filaments poses a challenge for their high-resolution structure determination. This review presents different approaches that have been used to study the structure and dynamics of T4aP and T2SS endopili by means of integrative structural biology, cryo-electron microscopy (cryo-EM), and molecular dynamics simulations. Their conserved features and differences are presented. The non-helical stretch in the long-conserved N-terminal helix which is characteristic of all members of the TFF and the impact of calcium on structure, function, and dynamics of these filaments are discussed in detail.
Asunto(s)
Microscopía por Crioelectrón , Fimbrias Bacterianas , Sistemas de Secreción Tipo II , Fimbrias Bacterianas/química , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Fimbrias Bacterianas/fisiología , Microscopía por Crioelectrón/métodos , Sistemas de Secreción Tipo II/química , Sistemas de Secreción Tipo II/metabolismo , Simulación de Dinámica Molecular , Conformación Proteica , Proteínas Fimbrias/química , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genéticaRESUMEN
Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process.
Asunto(s)
Microscopía por Crioelectrón , Fimbrias Bacterianas , Sistemas de Secreción Tipo IV , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Fimbrias Bacterianas/genética , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/química , Modelos Moleculares , Conjugación Genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Escherichia coli/metabolismo , Escherichia coli/genética , Conformación ProteicaRESUMEN
Amongst the major types of archaeal filaments, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). Within Sulfolobales, member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus system (Ups) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Ups in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility. Here, we present a cryoEM structure of the Aap of the archaeal model organism Sulfolobus acidocaldarius. We identify the component subunit as AapB and find that while its structure follows the canonical T4P blueprint, it adopts three distinct conformations within the pilus. The tri-conformer Aap structure that we describe challenges our current understanding of pilus structure and sheds new light on the principles of twitching motility.
Asunto(s)
Microscopía por Crioelectrón , Sulfolobus acidocaldarius , Sulfolobus acidocaldarius/metabolismo , Sulfolobus acidocaldarius/genética , Sulfolobus acidocaldarius/fisiología , Proteínas Arqueales/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Fimbrias Bacterianas/ultraestructura , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/fisiología , Fimbrias Bacterianas/química , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Modelos MolecularesRESUMEN
Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from small pilin proteins, can be many microns long and serve diverse functions, including adhesion, biofilm formation, motility, and intercellular communication. Here, we determine atomic structures of two distinct adhesive T4P from Saccharolobus islandicus via cryo-electron microscopy (cryo-EM). Unexpectedly, both pili were assembled from the same pilin polypeptide but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal T4P structures where all subunits are equivalent, whereas in the other filament, the tri-pilus, the same polypeptide exists in three different conformations. The three conformations in the tri-pilus are very different from the single conformation found in the mono-pilus, and involve different orientations of the outer immunoglobulin-like domains, mediated by a very flexible linker. Remarkably, the outer domains rotate nearly 180° between the mono- and tri-pilus conformations. Both forms of pili require the same ATPase and TadC-like membrane pore for assembly, indicating that the same secretion system can produce structurally very different filaments. Our results show that the structures of archaeal T4P appear to be less constrained and rigid than those of the homologous archaeal flagellar filaments that serve as helical propellers.
Asunto(s)
Proteínas Arqueales , Microscopía por Crioelectrón , Proteínas Fimbrias , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/química , Proteínas Fimbrias/ultraestructura , Proteínas Arqueales/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/ultraestructura , Modelos Moleculares , Fimbrias Bacterianas/ultraestructura , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/química , Conformación Proteica , Secuencia de AminoácidosRESUMEN
Caulobacter crescentus Tad (tight adherence) pili, part of the type IV pili family, are crucial for mechanosensing, surface adherence, bacteriophage (phage) adsorption, and cell-cycle regulation. Unlike other type IV pilins, Tad pilins lack the typical globular ß sheet domain responsible for pilus assembly and phage binding. The mechanisms of Tad pilus assembly and its interaction with phage ΦCb5 have been elusive. Using cryo-electron microscopy, we unveiled the Tad pilus assembly mechanism, featuring a unique network of hydrogen bonds at its core. We then identified the Tad pilus binding to the ΦCb5 maturation protein (Mat) through its ß region. Notably, the amino terminus of ΦCb5 Mat is exposed outside the capsid and phage/pilus interface, enabling the attachment of fluorescent and affinity tags. These engineered ΦCb5 virions can be efficiently assembled and purified in Escherichia coli, maintaining infectivity against C. crescentus, which presents promising applications, including RNA delivery and phage display.
Asunto(s)
Bacteriófagos , Caulobacter crescentus , Fimbrias Bacterianas , Caulobacter crescentus/citología , Caulobacter crescentus/metabolismo , Caulobacter crescentus/virología , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Microscopía por Crioelectrón , Bacteriófagos/química , Bacteriófagos/metabolismo , Proteínas Fimbrias , Escherichia coli , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
A dynamic field of study has emerged involving long-range electron transport by extracellular filaments in anaerobic bacteria, with Geobacter sulfurreducens being used as a model system. The interest in this topic stems from the potential uses of such systems in bioremediation, energy generation, and new bio-based nanotechnology for electronic devices. These conductive extracellular filaments were originally thought, based upon low-resolution observations of dried samples, to be type IV pili (T4P). However, the recently published atomic structure for the T4P from G. sulfurreducens, obtained by cryo-electron microscopy (cryo-EM), is incompatible with the numerous models that have been put forward for electron conduction. As with all high-resolution structures of T4P, the G. sulfurreducens T4P structure shows a partial melting of the α-helix that substantially impacts the aromatic residue positions such that they are incompatible with conductivity. Furthermore, new work using high-resolution cryo-EM shows that conductive filaments thought to be T4P are actually polymerized cytochromes, with stacked heme groups forming a continuous conductive wire, or extracellular DNA. Recent atomic structures of three different cytochrome filaments from G. sulfurreducens suggest that such polymers evolved independently on multiple occasions. The expectation is that such polymerized cytochromes may be found emanating from other anaerobic organisms.
Asunto(s)
Citocromos , Fimbrias Bacterianas , Geobacter , Nanocables , Nanocables/química , Nanocables/ultraestructura , Transporte de Electrón , Geobacter/química , Geobacter/metabolismo , Fimbrias Bacterianas/química , Fimbrias Bacterianas/ultraestructura , Citocromos/química , Citocromos/ultraestructura , Microscopía por CrioelectrónRESUMEN
Adhesive pili assembled through the chaperone-usher pathway are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria1-3. Archaic chaperone-usher pathway pili, the most diverse and widespread chaperone-usher pathway adhesins, are promising vaccine and drug targets owing to their prevalence in the most troublesome multidrug-resistant pathogens1,4,5. However, their architecture and assembly-secretion process remain unknown. Here, we present the cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii-a notorious multidrug-resistant nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into an ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed for the first time, to our knowledge, in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight multidrug-resistant bacterial infections.
Asunto(s)
Acinetobacter baumannii , Microscopía por Crioelectrón , Fimbrias Bacterianas , Chaperonas Moleculares , Acinetobacter baumannii/citología , Acinetobacter baumannii/ultraestructura , Elasticidad , Proteínas Fimbrias/química , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/ultraestructura , Fimbrias Bacterianas/química , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/ultraestructuraRESUMEN
Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F. tularensis, the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F. novicida. Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F. novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F. novicida.
Asunto(s)
Adhesión Bacteriana/genética , Proteínas Bacterianas , Fimbrias Bacterianas , Francisella , Tularemia , Factores de Virulencia , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Francisella/genética , Francisella/metabolismo , Francisella/patogenicidad , Francisella/ultraestructura , Francisella tularensis/genética , Francisella tularensis/metabolismo , Francisella tularensis/patogenicidad , Francisella tularensis/ultraestructura , Humanos , Ratones , Ratones Endogámicos BALB C , Tularemia/genética , Tularemia/metabolismo , Tularemia/patología , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/fisiología , Fimbrias Bacterianas/fisiología , Neisseria meningitidis/fisiología , Animales , Anticuerpos/uso terapéutico , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Línea Celular , Evaluación Preclínica de Medicamentos , Femenino , Fimbrias Bacterianas/química , Fimbrias Bacterianas/ultraestructura , Humanos , Infecciones Meningocócicas/tratamiento farmacológico , Ratones SCIDRESUMEN
The endospores (spores) of many Bacillus cereus sensu lato species are decorated with multiple hair/pilus-like appendages. Although they have been observed for more than 50 years, all efforts to characterize these fibers in detail have failed until now, largely due to their extraordinary resilience to proteolytic digestion and chemical solubilization. A recent structural analysis of B. cereus endospore appendages (Enas) using cryo-electron microscopy has revealed the structure of two distinct fiber morphologies: the longer and more abundant "Staggered-type" (S-Ena) and the shorter "Ladder-like" type (L-Ena), which further enabled the identification of the genes encoding the S-Ena. Ena homologs are widely and uniquely distributed among B. cereus sensu lato species, suggesting that appendages play important functional roles in these species. The discovery of ena genes is expected to facilitate functional studies involving Ena-depleted mutant spores to explore the role of Enas in the interaction between spores and their environment. Given the importance of B. cereus spores for the food industry and in medicine, there is a need for a better understanding of their biological functions and physicochemical properties. In this review, we discuss the current understanding of the Ena structure and the potential roles these remarkable fibers may play in the adhesion of spores to biotic and abiotic surfaces, aggregation, and biofilm formation.
Asunto(s)
Bacillus cereus/ultraestructura , Proteínas Bacterianas/química , Fimbrias Bacterianas/ultraestructura , Esporas Bacterianas/ultraestructura , Bacillus cereus/genética , Bacillus cereus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biopelículas/crecimiento & desarrollo , Microscopía por Crioelectrón , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismoRESUMEN
Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that class 1a pili of uropathogenic E. coli (UPEC), as well as class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC class 5 pili. Examining structural and steered molecular dynamics simulation data, we find that this difference in class 1 pili subunit behavior originates from an α-helical motif that can unfold when exposed to force. A disulfide bond cross-linking ß-strands in class 1 pili stabilizes subunits, allowing them to tolerate higher forces than class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche, since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of class 5 ETEC pili.
Asunto(s)
Adhesinas de Escherichia coli/química , Escherichia coli Enterotoxigénica/ultraestructura , Proteínas Fimbrias/química , Fimbrias Bacterianas/ultraestructura , Escherichia coli Uropatógena/ultraestructura , Adhesinas de Escherichia coli/genética , Adhesinas de Escherichia coli/metabolismo , Adhesión Bacteriana , Sitios de Unión , Fenómenos Biomecánicos , Cisteína/química , Cisteína/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Expresión Génica , Cinética , Simulación de Dinámica Molecular , Pinzas Ópticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Termodinámica , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/metabolismoRESUMEN
Bacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here, we isolate endospore appendages (Enas) from the food poisoning outbreak strain B. cereus NVH 0075-95 and find proteinaceous fibers of two main morphologies: S- and L-Ena. By using cryoEM and 3D helical reconstruction of S-Enas, we show these to represent a novel class of Gram-positive pili. S-Enas consist of single domain subunits with jellyroll topology that are laterally stacked by ß-sheet augmentation. S-Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought, and chemical damage. Phylogenomic analysis reveals a ubiquitous presence of the ena-gene cluster in the B. cereus group, which include species of clinical, environmental, and food importance. We propose Enas to represent a new class of pili specifically adapted to the harsh conditions encountered by bacterial spores.
Asunto(s)
Bacillus cereus/ultraestructura , Proteínas Bacterianas/química , Fimbrias Bacterianas/ultraestructura , Bacillus cereus/genética , Proteínas Bacterianas/genética , Microscopía por Crioelectrón , Fimbrias Bacterianas/química , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Estabilidad ProteicaRESUMEN
The bacterial flagellum consists of a long extracellular filament that is rotated by a motor embedded in the cell envelope. While flagellar assembly has been extensively studied,1 the disassembly process remains less well understood. In addition to the programmed flagellar ejection that occurs during the life cycle of Caulobacter crescentus, we and others have recently shown that many bacterial species lose their flagella under starvation conditions, leaving relic structures in the outer membrane.2-7 However, it remains unknown whether the programmed flagellar ejection of C. crescentus leaves similar relics or not. Here, we imaged the various stages of the C. crescentus life cycle using electron cryo-tomography (cryo-ET) and found that flagellar relic subcomplexes, akin to those produced in the starvation-induced process, remain as a result of flagellar ejection during cell development. This similarity suggests that the programmed flagellar ejection of C. crescentus might share a common evolutionary path with the more general, and likely more ancient,3 starvation-related flagellar loss.
Asunto(s)
Caulobacter crescentus/fisiología , Pared Celular/metabolismo , Flagelos/fisiología , Lipopolisacáridos/metabolismo , Peptidoglicano/metabolismo , Cuerpos Basales/fisiología , Cuerpos Basales/ultraestructura , Caulobacter crescentus/metabolismo , Caulobacter crescentus/ultraestructura , Pared Celular/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Fimbrias Bacterianas/fisiología , Fimbrias Bacterianas/ultraestructura , Flagelos/ultraestructuraRESUMEN
Long-distance extracellular electron transfer has been observed in Gram-negative bacteria and plays roles in both natural and engineering processes. The electron transfer can be mediated by conductive protein appendages (in short unicellular bacteria such as Geobacter species) or by conductive cell envelopes (in filamentous multicellular cable bacteria). Here we show that Lysinibacillus varians GY32, a filamentous unicellular Gram-positive bacterium, is capable of bidirectional extracellular electron transfer. In microbial fuel cells, L. varians can form centimetre-range conductive cellular networks and, when grown on graphite electrodes, the cells can reach a remarkable length of 1.08 mm. Atomic force microscopy and microelectrode analyses suggest that the conductivity is linked to pili-like protein appendages. Our results show that long-distance electron transfer is not limited to Gram-negative bacteria.
Asunto(s)
Transporte de Electrón/fisiología , Bacterias Grampositivas/metabolismo , Bacillaceae/citología , Bacillaceae/crecimiento & desarrollo , Bacillaceae/metabolismo , Fuentes de Energía Bioeléctrica/microbiología , Conductividad Eléctrica , Electrodos/microbiología , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Bacterias Grampositivas/citología , Bacterias Grampositivas/crecimiento & desarrollo , Grafito , Microscopía de Fuerza Atómica , NanocablesRESUMEN
The ability to form biofilms is shared by many microorganisms, including archaea. Cells in a biofilm are encased in extracellular polymeric substances that typically include polysaccharides, proteins, and extracellular DNA, conferring protection while providing a structure that allows for optimal nutrient flow. In many bacteria, flagella and evolutionarily conserved type IV pili are required for the formation of biofilms on solid surfaces or floating at the air-liquid interface of liquid media. Similarly, in many archaea it has been demonstrated that type IV pili and, in a subset of these species, archaella are required for biofilm formation on solid surfaces. Additionally, in the model archaeon Haloferax volcanii, chemotaxis and AglB-dependent glycosylation play important roles in this process. H. volcanii also forms immersed biofilms in liquid cultures poured into petri dishes. This study reveals that mutants of this haloarchaeon that interfere with the biosynthesis of type IV pili or archaella, as well as a chemotaxis-targeting transposon and aglB deletion mutants, lack obvious defects in biofilms formed in liquid cultures. Strikingly, we have observed that these liquid-based biofilms are capable of rearrangement into honeycomb-like patterns that rapidly form upon removal of the petri dish lid, a phenomenon that is not dependent on changes in light or oxygen concentration but can be induced by controlled reduction of humidity. Taken together, this study demonstrates that H. volcanii requires novel, unidentified strategies for immersed liquid biofilm formation and also exhibits rapid structural rearrangements.IMPORTANCE This first molecular biological study of archaeal immersed liquid biofilms advances our basic biological understanding of the model archaeon Haloferax volcanii Data gleaned from this study also provide an invaluable foundation for future studies to uncover components required for immersed liquid biofilms in this haloarchaeon and also potentially for liquid biofilm formation in general, which is poorly understood compared to the formation of biofilms on surfaces. Moreover, this first description of rapid honeycomb pattern formation is likely to yield novel insights into the underlying structural architecture of extracellular polymeric substances and cells within immersed liquid biofilms.
Asunto(s)
Biopelículas , Proteínas Fimbrias/metabolismo , Haloferax volcanii/metabolismo , Proteínas Fimbrias/genética , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Glicosilación , Haloferax volcanii/citología , Haloferax volcanii/genética , Polisacáridos/metabolismoRESUMEN
Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation.
Asunto(s)
Sistemas de Secreción Bacterianos/ultraestructura , Microscopía por Crioelectrón , Fimbrias Bacterianas/ultraestructura , Secretina/química , Vibrio cholerae/metabolismo , Vibrio cholerae/ultraestructura , Cisteína/genética , Proteínas de la Membrana/ultraestructura , Modelos Moleculares , Mutación/genética , Filogenia , Dominios Proteicos , Transformación BacterianaRESUMEN
Extracellular electron transfer via filamentous protein appendages called 'microbial nanowires' has long been studied in Geobacter and other bacteria because of their crucial role in globally-important environmental processes and their applications for bioenergy, biofuels, and bioelectronics. Thousands of papers thought these nanowires as pili without direct evidence. Here, we summarize recent discoveries that could help resolve two decades of confounding observations. Using cryo-electron microscopy with multimodal functional imaging and a suite of electrical, biochemical, and physiological studies, we find that rather than pili, nanowires are composed of cytochromes OmcS and OmcZ that transport electrons via seamless stacking of hemes over micrometers. We discuss the physiological need for two different nanowires and their potential applications for sensing, synthesis, and energy production.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citocromos/metabolismo , Fimbrias Bacterianas/metabolismo , Bacterias Gramnegativas/metabolismo , Proteínas Bacterianas/ultraestructura , Citocromos/ultraestructura , Transporte de Electrón , Fimbrias Bacterianas/ultraestructura , Geobacter/metabolismo , Geobacter/ultraestructura , Bacterias Gramnegativas/ultraestructura , Modelos Moleculares , Nanocables/ultraestructuraRESUMEN
Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ('wide' and 'narrow'), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.
Asunto(s)
Fimbrias Bacterianas/química , Fimbrias Bacterianas/ultraestructura , Thermus thermophilus/ultraestructura , Microscopía por Crioelectrón , ADN/metabolismo , Proteínas Fimbrias/química , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Espectrometría de Masas , Modelos Moleculares , Estructura Secundaria de Proteína , Thermus thermophilus/química , Thermus thermophilus/metabolismoRESUMEN
The type II secretion system (T2SS) is a multiprotein envelope-spanning assembly that translocates a wide range of virulence factors, enzymes and effectors through the outer membrane of many Gram-negative bacteria1-3. Here, using electron cryotomography and subtomogram averaging methods, we reveal the in vivo structure of an intact T2SS imaged within the human pathogen Legionella pneumophila. Although the T2SS has only limited sequence and component homology with the evolutionarily related type IV pilus (T4P) system4,5, we show that their overall architectures are remarkably similar. Despite similarities, there are also differences, including, for example, that the T2SS-ATPase complex is usually present but disengaged from the inner membrane, the T2SS has a much longer periplasmic vestibule and it has a short-lived flexible pseudopilus. Placing atomic models of the components into our electron cryotomography map produced a complete architectural model of the intact T2SS that provides insights into the structure and function of its components, its position within the cell envelope and the interactions between its different subcomplexes.