Fluorescence assays for F-pili and their application

Abstract

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...

F-pili dynamics by live-cell imaging

“…We showed that fluorescent R17 bacteriophage binding to fixed E. coli Fϩ cells could be used to identify F-pili (23). We have now extended this approach to visualize filament dynamics on live cells.…”

“…E. coli K-12 strains HfrH and AE2386/JCFL0 were from the authors' strain collection and have been described (23). The strains expressed the cytoplasmic, IPTG-inducible fluorescent marker DsRed Express (31).…”

The Structure of F-Pili

“…F-pili were purified from the supernatant fraction by two cycles of isopycnic sedimentation in sucrose gradients (density range = 1.16 to 1.29 g/mL). Fractions at ∼1.2 g/mL were examined by fluorescence microscopy for F-pili28 and by SDS gel electrophoresis for purity (where preparations were judged to be >90% F-pilin). Images of frozen-hydrated F-pili were collected on a Tecnai 20 field emission gun microscope at 200 keV.…”

Common Requirement for the Relaxosome of Plasmid R1 in Multiple Activities of the Conjugative Type IV Secretion System

“…The titer was 1.02 ϫ 10 14 PFU/ml. Phage were labeled with Alexa Fluor 488 (Invitrogen) as described previously (55), except that phages were harvested from a discontinuous CsCl gradient at a of ϳ1.45 g/ml and the addition of glycerol was omitted. After labeling, phage were treated with chloroform to dissolve detached R1-16 pili still present in the preparation, dialyzed in P buffer, and then filtered through a 0.2-m filter and stored at 4°C.…”