Mutational analysis of F‐pilin reveals domains for pilus assembly, phage infection and DNA transfer

Manchak, Jan; Anthony, Greenberg; Frost, Laura S.

doi:10.1046/j.1365-2958.2002.02731.x

Cited by 56 publications

(57 citation statements)

References 36 publications

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“…This can be misleading. As we have shown here for the G69C F-pilin mutant, and as Grossman & Silverman (1989) and Manchak et al (2002) have also shown, altered F-pilins can be incorporated into filaments that fail to bind RNA bacteriophage. Such ambiguities might be minimized by using cysteine-reactive fluorescent dyes, rather than fluorescent bacteriophage, in conjunction with cysteine-containing F-pili.…”

Section: Discussionsupporting

confidence: 73%

“…The effects of the G69C mutation can not be attributed to the presence of cysteine at this locus since the G69D mutation had much the same effects . Interestingly, neither we nor Manchak et al (2002), nor the more limited study by , identified F-pilin missense mutants that significantly reduced Ff bacteriophage sensitivity and DNA donor activity without also reducing or abolishing RNA bacteriophage sensitivity. One explanation for these data is that donor activity and DNA bacteriophage sensitivity are relatively robust functions with respect to modest alterations to F-pilin structure, whereas RNA bacteriophage infection is more sensitive.…”

Section: Discussionmentioning

confidence: 60%

“…Such ambiguities might be minimized by using cysteine-reactive fluorescent dyes, rather than fluorescent bacteriophage, in conjunction with cysteine-containing F-pili. Manchak et al (2002), examining the effects of single missense mutations of F-pilin, found that in general DNA donor activity and sensitivity to filamentous DNA bacteriophage tracked together. In contrast, several mutations abolished RNA bacteriophage sensitivity with less of an effect on the other two functions.…”

Section: Discussionmentioning

confidence: 99%

“…Similar to studies by Manchak et al (2002), we have been constructing and characterizing F-pilin missense mutants, in conjunction with structural studies of F-pili necessary to interpret their effects. Here, we use a subset of such mutants to illustrate the utility and one limitation of the methods described above.…”

Section: Cysteine-containing F-pilin Mutantsmentioning

confidence: 99%

“…Plasmid DNA was isolated and traA inserts sequenced in both directions. Following Manchak et al (2002), we transferred the mutant and wild-type traA genes into the vector pBAD/Myc-His A (Invitrogen). These plasmids were introduced by transformation into TOP10/pOX38traA : : cat.…”

mentioning

confidence: 99%

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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: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Section: Cysteine-containing F-pilin Mutantsmentioning

confidence: 99%

mentioning

confidence: 99%

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Fluorescence assays for F-pili and their application

et al. 2005

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Investigation of Bacteriophage MS2 Viral Dynamics Using Model Discrimination Analysis and the Implications for Phage Therapy

Jain¹,

Knorr²,

Bernacki³

et al. 2006

Biotechnol Progress

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Lytic phages infect their bacterial hosts, use the host machinery to replicate, and finally lyse and kill their hosts, releasing progeny phages. Various mathematical models have been developed that describe these phage-host viral dynamics. The aim of this study was to determine which of these models best describes the viral dynamics of lytic RNA phage MS2 and its host Escherichia coli C-3000. Experimental data consisted of uninfected and infected bacterial cell densities, free phage density, and substrate concentration. Parameters of various models were either determined directly through other experimental techniques or estimated using regression analysis of the experimental data. The models were evaluated using a Bayesian-based model discrimination technique. Through model discrimination it was shown that phage-resistant cells inhibited the growth of phage population. It was also shown that the uninfected bacterial population was a quasispecies consisting of phage-sensitive and phage-resistant bacterial cells. When there was a phage attack the phage-sensitive cells died out and the phage-resistant cells were selected for and became the dominant strain of the bacterial population.

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Plasmid Molecular Biology

Bacterial and Bacteriophage Genetics

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Mutational analysis of F‐pilin reveals domains for pilus assembly, phage infection and DNA transfer

Cited by 56 publications

References 36 publications

Fluorescence assays for F-pili and their application

Fluorescence assays for F-pili and their application

Investigation of Bacteriophage MS2 Viral Dynamics Using Model Discrimination Analysis and the Implications for Phage Therapy

Plasmid Molecular Biology

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