Academic literature on the topic 'Competence pilus'

ABSTRACT Thermus thermophilus HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. To identify genes of the natural transformation machinery of T. thermophilus HB27, we performed homology searches in the partially completed T. thermophilus genomic sequence for conserved competence genes. These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria. Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency. One competence protein is similar to DprA of Haemophilus influenzae, seven are similar to type IV pilus proteins of Pseudomonas aeruginosa or Neisseria gonorrhoeae (PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in Deinococcus radiodurans R1. Analysis of the piliation phenotype of T. thermophilus HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent pil mutants of Thermus, with the exception of the pilF mutant, were devoid of pilus structures. These results suggest that pili and natural transformation in T. thermophilus HB27 are functionally linked.

ABSTRACT The gonococcal pilus is a major virulence factor that has well-established roles in mediating epithelial cell adherence and DNA transformation. Gonococci expressing four gonococcal pilin variants with distinct piliation properties under control of the lac regulatory system were grown in different levels of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). These pilin variants expressed various levels of pilin message and pilin protein in response to the level of IPTG in the growth medium. Moreover, posttranslational modifications of the variant pilin proteins were detected, including S-pilin production and glycosylation. The ratio of the modified and unmodified pilin forms did not substantially change with different levels of pilin expression, showing that these modifications are not linked to pilin expression levels. DNA transformation competence was also influenced by IPTG levels in the growth medium. Substantial increases in transformation competence over an isogenic, nonpiliated mutant were observed when limited amounts of three of the pilin variants were expressed. Immunoelectron microscopy showed that when limited amounts of pilin are expressed, pili are rare and do not explain the pilin-dependent transformation competence. This pilin-dependent transformation competence required prepilin processing, the outer membrane secretin PilQ, and the twitching-motility-regulating protein PilT. These requirements show that a fully functional pilus assembly apparatus is required for DNA uptake when limited pilin is produced. We conclude that the pilus assembly apparatus functions to import DNA into the bacterial cell in a pilin-dependent manner but that extended pili are not required for transformation competence.

ABSTRACT The ubiquitous species Pseudomonas stutzeri has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termedpilT. The deduced amino acid sequence has identity with PilT of Pseudomonas aeruginosa (94%), Neisseria gonorrhoeae (67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [3H]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of pilT a gene, termedpilU, coding for a putative protein with 88% amino acid identity with PilU of P. aeruginosa was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable P. aeruginosa. When thepilAI gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in pilAI::Gmr cells but allowed transformability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the pilT mutation. Considering the hypothesized role of pilT + in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the pilT-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.

ABSTRACT The extreme thermophile Thermus thermophilus HB27 exhibits high frequencies of natural transformation. Although we recently reported identification of the first competence genes in Thermus, the molecular basis of DNA uptake is unknown. A pilus-like structure is assumed to be involved. Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of T. thermophilus. Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (pilA1 to pilA4), is essential for natural transformation. Additionally, comZ, a new competence gene with no similarity to known genes, was identified. Analysis of the piliation phenotype revealed wild-type piliation of a pilA1-pilA3Δkat mutant and a comZ mutant, whereas a pilA4 mutant was found to be completely devoid of pilus structures. These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the T. thermophilus pilus structures are type IV pili. Furthermore, the loss of the transformation and piliation phenotype in the pilA4 mutant suggests that type IV pili are implicated in natural transformation of T. thermophilus HB27.

Transformation is a widespread mechanism of horizontal gene transfer in bacteria. DNA uptake to the periplasmic compartment requires a DNA-uptake pilus and the DNA-binding protein ComEA. In the gram-negative bacteria, DNA is first pulled toward the outer membrane by retraction of the pilus and then taken up by binding to periplasmic ComEA, acting as a Brownian ratchet to prevent backward diffusion. A similar mechanism probably operates in the gram-positive bacteria as well, but these systems have been less well characterized. Transport, defined as movement of a single strand of transforming DNA to the cytosol, requires the channel protein ComEC. Although less is understood about this process, it may be driven by proton symport. In this review we also describe various phenomena that are coordinated with the expression of competence for transformation, such as fratricide, the kin-discriminatory killing of neighboring cells, and competence-mediated growth arrest.

ABSTRACT The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolateE. corrodens VA1. Earlier work defined the pilAlocus which includes pilA1, pilA2,pilB, and hagA. Both pilA1 andpilA2 predict a type IV pilin, whereas pilBpredicts a putative pilus assembly protein. The role ofhagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilAmutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated forpilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation ofhagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.

Neisseria meningitidis, a causative agent of meningitis and septicaemia, expresses type IV pili, a feature correlating with the uptake of exogenous DNA from the environment by natural transformation. The outer membrane complex PilQ, through which pili are extruded and retracted, has previously been shown to bind DNA in its pore region. In order to further elucidate how DNA is transported across the membranes, we searched for DNA binding proteins within the meningococcal inner membrane. Inner membrane fractions from a panel of neisserial strains were subjected to a solid-phase overlay assay with DNA substrates, and MS was subsequently employed to identify proteins that bind DNA. A number of DNA binding components were detected, including the pilus biogenesis component PilG, the competence protein ComL, and the cell division ATP-binding protein FtsE, as well as two hypothetical proteins. The DNA binding activity of these components was not dependent on the presence of the neisserial DNA uptake sequence. Null mutants, corresponding to each of the proteins identified, were constructed to assess their phenotypes. Only mutants defective in pilus biogenesis were non-competent and non-piliated. The DNA binding activity of the pilus biogenesis components PilQ and PilG and the phenotypes of their respective null mutants suggest that these proteins are directly involved as players in natural transformation, and not only indirectly, through pilus biogenesis.

Nombreuses espèces bactériennes présentent des fibres à leur surface qui leur permettent d’interagir avec leur environnement. Les pili de type 4 (PT4) sont des fibres longues, fines et flexibles, impliquées dans des fonctions multiples telles que l'adhérence, la motilité, la sécrétion, l'import d'ADN et la formation des biofilms. Ils sont composés de milliers de copies de sous-unité majeure de piline et sont assemblés par un complexe protéique localisé dans l'enveloppe bactérienne. Dans cette étude, nous nous sommes intéressés aux PT4 chez Escherichia coli entérohémorragique (EHEC) de sérotype O157: H7. EHEC est un agent pathogène humain d'origine alimentaire qui provoque des épidémies de diarrhées hémorragiques et/ou des atteintes rénales sévères appelées syndrome hémolytique et urémique (SHU), qui peuvent être mortelles. Les PT4 chez EHEC sont composés de la sous-unité majeure PpdD et probablement des pilines mineures PpdA, PpdB, YgdB et PpdC en plus faible abondance.Dans cette étude notre objectif était de décrire la structure des PT4 chez EHEC, en tant que modèle de la famille des pili conservés chez les entérobactéries. La structure est indispensable pour décrire le mode d'action de ces organites. Dans cette étude, nous avons abordé la structure de l'EHEC T4P ainsi que les bases moléculaires de leur assemblage. En raison de la nature flexible et non covalente des fibres T4P, nous avons utilisé une approche intégrative incluant des données obtenues par cryo-microscopie électronique (cryo-ME), spectroscopie RMN, modélisation moléculaire et analyses biochimiques pour déterminer la structure des pili PpdD. Les interactions entre les sous-unités de pilines présentes dans la fibre ont été étudiées par mutagenèse dirigée et analyses fonctionnelles. Ces expériences nous ont permis d’identifier les résidus de PpdD essentiels pour l’assemblage des pili.Les T4aP ont été peu étudiés chez les entérobactéries. Bien que tous les gènes nécessaires soient présents chez E. coli, les conditions induisant leur expression restent inconnues. Cependant, les gènes codant pour les PT4 chez E. coli sont co-régulés avec des gènes codant pour la machinerie impliquée dans l’import d'ADN, ce qui suggère leur rôle dans la compétence naturelle. Afin de faciliter l'étude de PT4, nous avons réalisé une reconstitution fonctionnelle de l'EHEC T4PS chez E. coli K-12 non pathogène par l'expression contrôlée des gènes de PT4 clonés dans un opéron artificiel. Cette construction nous a permis de réaliser des analyses comparatives d'assemblage des pili par le système hétérologue (système de sécrétion de type 2 provenant d'une autre entérobactérie, Klebsiella oxytoca) et le système d'assemblage propre aux PT4 chez EHEC. Leur analyse par cryo-ME a montré que les pili assemblés sont identiques, indiquant ainsi que ce sont les pilines majeures qui déterminent la structure et la symétrie des fibres. Les résultats des études comparatives ont également apporté des informations importantes sur l'assemblage des pili. Nous avons identifié des composants de la machinerie d'assemblage qui interagissent avec la piline majeure PpdD. La formation des dimères de PpdD et ses interactions spécifiques avec les facteurs d’assemblage semblent importantes pour la stabilité de la piline avant l'assemblage en pilus. L’ensemble de nos résultats consitutent des bases pour de futures analyses structurales et fonctionnelles des pili chez les entérobactéries
Many bacterial species display surface fibers to interact with the surrounding environment. Type 4 pili (T4P) are long and thin, flexible fibers involved in a variety of functions including adherence, motility, secretion, DNA uptake and biofilm formation. They are composed of thousands of copies of major pilin subunits and are assembled by a protein complex localized in the bacterial envelope. In this study we focused on the T4P from Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7. EHEC is a human food­borne pathogen that causes outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS), which can lead to a lethal outcome. EHEC T4P is composed of the major subunit PpdD and presumably in lower abundance by minor pilins PpdA, PpdB, YgdB and PpdC.This study has aimed to describe the structure of the T4P from EHEC, a representative of pili conserved in enterobacteria. Structural information provides insights that can relate to the mode of action of these organelles. In this project we addressed the structure of the EHEC T4P and the molecular basis of their assembly. Due to the flexible and non-covalent nature of T4P fibers we used an integrative approach to combine information obtained by cryo-electron microscopy, NMR spectroscopy, molecular modeling and biochemical analyses to obtain a high quality structure of the EHEC T4P. In addition, from functional, interaction and mutational analysis we gained insights into the molecular interactions between the pilin subunits present in the fiber. The T4P are poorly studied in enterobacterial systems; despite the presence of all the necessary genes, the conditions that trigger their expression in E. coli are unknown. In addition, in E. coli the T4P-related genes are coregulated with genes encoding the DNA uptake machinery, suggesting their role in natural competence. In order to facilitate the study of T4P, we achieved a functional reconstitution of the EHEC T4PS in the non-pathogenic E. coli K-12 through the controlled expression of the T4P genes cloned together as a single artificial operon. With this accomplishment we were able to perform comparative analyses between pili assembled by a heterologous system (the Type 2 secretion system from another enterobacterium, Klebsiella oxytoca) and by the cognate EHEC T4P assembly system. CryoEM analysis showed that these pili are identical, indicating that major pilins are the key determinants of fiber structure and symmetry. The results also led us to obtain important insights into pilus assembly and to characterize PpdD interactions with the assembly machinery. These interactions and PpdD dimerization were required for pilin stability prior to pilus assembly, highlighting important early steps involving targeting of subunits to the assembly machinery. Together, these results lay the foundations for future structural and functional studies of enterobacterial T4P

Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of small macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of cryo-EM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Of our seven designs, the best construct used a designed ankyrin repeat protein (DARPin) rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target: GFP. We solved the structure of this complex to 3.0 Å resolution overall, with 5-8 Å resolution in the GFP region. As flexibility in the DARPin position limited the overall resolution of the target, we describe strategies to rigidify this element.

Natural competence is the process by which bacteria take up genetic material from their environment and integrate it into their genome using homologous recombination. In Vibrio cholerae, the Type IV competence pilus is thought to mediate DNA uptake by binding DNA and retracting back toward the cell. How the DNA enters the periplasm is unclear. One hypothesis suggests that the DNA-bound Type IV competence pilus retracts completely so that the DNA would pass through the outer membrane secretin pore (PilQ). PilQ is a 870 kDa outer membrane pore with C14 symmetry. Here, we purify the V. cholerae PilQ secretin from V. cholerae cells in amphipols for single particle cryogenic electron microscopy (cryoEM). We solve the structure to 3.0 Å and provide insight on the channel DNA may traverse through during uptake.

El informe hace referencia a la aplicación del modelo matemático de "espacio de estados" a un rodal de Pinus radiata en Chile. Esta metodología es considerada un sistema dinámico, cuyo estado en un momento determinado "t, p(t)" puede ser representado por variables de estado cuidadosamente seleccionadas. Este estado evoluciona con el tiempo en un espacio, el cual abarca todos los posibles estados que el sistema pueda tener. Ocupando la notación y los conceptos de esta aproximación en las Ciencias Forestales, el rodal es considerado como un sistema. Esto significa que es una colección de elementos: los árboles, relaciones entre ellos, competencia por luz, agua, y nutrientes y las interacciones con el medio ambiente.

This chapter looks closely at John Bunyan’s first major narrative, his conversion account Grace Abounding to the Chief of Sinners (1666), exploring interwoven qualities of intense religious confession, especially the deep sense of sinful guilt; metaphorical vitality drawn from Bunyan’s local experience in Bedfordshire; animated, personified, medicinal citation of the Bible (and other pious books); modes of social abjection; and pursuit of the authority to speak. Grace Abounding was composed when Bunyan was in prison under the terms of the 1664 Conventicle Act, and hence he was using the conversion narrative as a kind of published sermon. Later revisions reduced or erased traces of his earlier religious radicalism, and demonstrated his growing competence as minister and writer, but sometimes compromised authentically remembered spontaneous experience.

Hundreds of Qur’anic verses pertain to women and girl figures. These figures play pivotal roles in Islamic sacred history, and the Qur’an celebrates the aptitudes of many such figures in the realms of spirituality, politics, and family. Some women are political adversaries of prophets or use their agency in morally corrupt ways; however, the Qur’an presents many more examples of pious women and girls, including those who birth, protect, guide, and inspire prophets. This book outlines how female figures—old, young, barren, fertile, chaste, profligate, saintly, and reproachable—enter Islamic sacred history and advance the Qur’an’s overarching didactic aims. The analysis considers all the major and minor female figures referenced in the Qur’an, including those who appear in narratives of sacred history, in parables, in verses that allude to events contemporaneous with the Qur’an, and in descriptions of the eternal abode. Female personalities appear in the Qur’anic accounts of human origins, in stories of the founding and destruction of nations, and in narratives of conquest, filial devotion, romantic attraction, and more. This work gives attention to these wide-ranging depictions and to themes related to sexual relations, kinship relations, divine-human relationships, female embodiment, and women’s social roles. Analysis focuses on lexical features of the Qur’an, intra-textual resonances, and thematic juxtapositions. The book explores Qur’anic dictates involving gender relations and highlights female spiritual competencies.