Fluorescence assays for F-pili and their application.
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.
Daehnel
Katrin
K
Program in Cell, Molecular, and Developmental Biology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.
Harris
Robin
R
Program in Cell, Molecular, and Developmental Biology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.
Maddera
Lucinda
L
Program in Cell, Molecular, and Developmental Biology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.
Silverman
Philip
P
Program in Cell, Molecular, and Developmental Biology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.
D000595:Amino Acid Sequence; D003227:Conjugation, Genetic; D003545:Cysteine; D048168:Escherichia coli K12 / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000821:virology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005144:F Factor / Q000235:genetics*; D036781:Fimbriae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation; D012317:RNA Phages / Q000378:metabolism* / Q000502:physiology
D029968:Escherichia coli Proteins; C444040:F pilin, E coli; D005456:Fluorescent Dyes; D036781:Fimbriae Proteins; D003545:Cysteine