Light-microscopic Visualization of F and Type 1 Pili

Biebricher, Christof Κ.; DÜker, Eva-Maria

doi:10.1099/00221287-130-4-941

Cited by 8 publications

(5 citation statements)

References 26 publications

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“…Only the 37 kDa protein, which corresponds in size to that of the bacteriophage A or maturation protein (Weber & Konigsberg, 1975), contained traces of fluorescence. Biebricher & Duker (1984) first showed that fluorescent RNA bacteriophage bound to F-pili could be visualized by fluorescence microscopy. The result of a similar experiment but carried out with current imaging and image processing technology is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Fluorescence assays for F-pili and their application

et al. 2005

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Conjugative pili are extracellular filaments elaborated by Gram-negative bacteria expressing certain type IV secretion systems. They are required at the earliest stages of conjugal DNA transfer to establish specific and secure cell-cell contacts. Conjugative pili also serve as adsorption organelles for both RNA and DNA bacteriophages. Beyond these facts, the structure, formation and function of these filaments are poorly understood. This paper describes a rapid, quantitative assay for F-pili encoded by the F plasmid type IV secretion system. The assay is based on the specific lateral adsorption of icosahedral RNA bacteriophage R17 by F-pili. Bacteriophage particles conjugated with a fluorescent dye, Alexa 488, and bound to F-pili defined filaments visible by immunofluorescence microscopy. F-pili attached to F + cells and free F-pili were both visible by this method. For quantification, cell-bound bacteriophage were separated from free bacteriophage particles by sedimentation and released by suspending cell pellets in 0?1 % SDS. Fluorescence in cell-free supernatant fractions was measured by fluorometry. The authors present a characterization of this assay and its application to F-pilus formation by cells carrying mutations in the gene for the F-pilus subunit F-pilin. Each mutation introduced a cysteine, which F-pilin normally lacks, at a different position in its primary structure. Cysteine residues in the N-terminal domain I abolished filament formation as measured by fluorescent R17 binding. This was confirmed by measurements of DNA donor activity and filamentous DNA bacteriophage infection. With one exception (G53C), cysteines elsewhere in the F-pilin primary structure did not abolish filament formation, although some mutations differentially affected F-pilus functions. INTRODUCTIONConjugative pili are characteristic of type IV secretion systems that mediate horizontal gene transfer by Gramnegative bacteria (Valentine et al., 1969;Ippen-Ihler & Maneewannakul, 1991;Fullner et al., 1996;Lai & Kado, 1998). Insofar as they have been examined, these extracellular filaments are repeats of one quantitatively predominant subunit Lai & Kado, 1998; Eisenbrandt et al., 1999). Conjugative pili and the corresponding pilin subunits are generally identified by the origin of the type IV secretion system of which they are components. The present report concerns F-pili, which are composed of F-pilin and elaborated by F + strains of Escherichia coli.F-pili are 8-9 nm in diameter and of indeterminate length. Structurally, they are hollow cylinders with a hydrophilic axial lumen that is accessible to the aqueous medium (Folkhard et al., 1979;Silverman, 1997). Functionally, F-pili make initial, SDS-sensitive contacts between donor and recipient cells (Achtman & Skurray, 1977;Manning & Achtman, 1979). Thereafter, F-pili retract so that donor and recipient cells are in direct surface-tosurface contact (Durrenberger et al., 1991). DNA transfer between closely apposed cells appears to be general (Samuels et al., 2000;Lawley et al., 20...

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Section: Resultsmentioning

confidence: 99%

Fluorescence assays for F-pili and their application

et al. 2005

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“…In fact, it has been established experimentally that a minor fraction of bacteriophage Qb population of only 0.7 ± 1.6 % contained infectious particles. [6] With eukaryotic picorna viruses, the relation of infectious to total virus particles even decreases to the level of 10 À3 ± 10 À4 . [7] These findings suggest that virus genomes cannot tolerate many additional mutations without substantially losing their viability.…”

Section: Introductionmentioning

confidence: 99%

An Error-Prone T7 RNA Polymerase Mutant Generated by Directed Evolution

Brakmann

Grzeszik

2001

ChemBioChem

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Viruses replicate their genomes at exceptionally high mutation rates. Their offspring evolve rapidly and therefore, are able to evade common immunological and chemical antiviral agents. In parallel, virus genomes cannot tolerate a further increase in mutation rate: Experimental evidence exists that even few additional mutations are sufficient for the extinction of a viral population. A future antiviral strategy might therefore aim at increasing the error-producing capacity of viral replication enzymes. We employed the principles of directed evolution and developed a scheme for the stringent positive selection of error-prone polymerase activity. A mutant T7 RNA polymerase with a nucleotide substitution error rate at least 20-fold greater than that of the wild-type was selected. This enzyme synthesized highly heterogeneous RNA products in vitro or in vivo and also decreased the replication efficiency of wild-type bacteriophage T7 during infection.

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“…Conclusively, the study of viral entry does not only address questions about complexity of viral mechanisms, it also helps to gain insight into composition and functioning of the bacterial and archaeal cell surface [43,102,103].…”

Section: Discussionmentioning

confidence: 99%

Viral Hijack of Filamentous Surface Structures in Archaea and Bacteria

Tittes

Schwarzer

Quax

2021

Viruses

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The bacterial and archaeal cell surface is decorated with filamentous surface structures that are used for different functions, such as motility, DNA exchange and biofilm formation. Viruses hijack these structures and use them to ride to the cell surface for successful entry. In this review, we describe currently known mechanisms for viral attachment, translocation, and entry via filamentous surface structures. We describe the different mechanisms used to exploit various surface structures bacterial and archaeal viruses. This overview highlights the importance of filamentous structures at the cell surface for entry of prokaryotic viruses.

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Light-microscopic Visualization of F and Type 1 Pili

Cited by 8 publications

References 26 publications

Fluorescence assays for F-pili and their application

Fluorescence assays for F-pili and their application

An Error-Prone T7 RNA Polymerase Mutant Generated by Directed Evolution

Viral Hijack of Filamentous Surface Structures in Archaea and Bacteria

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