Kinetic Studies of the Interaction between MS2 Phage and F Pilus of Escherichia coli

Abstract

The kinetics of the binding reaction of MS2 phage to free F pili, which were highly purified from Escherichia coli, has been studied using a membrane filter assay. The rate of dissociation ( k d ) of the MS2-phage -F-pilus complex is very slow and follows first-order kinetics with a half-life of 4.2 h at 30°C in the standard buffer. The dissociation rate is rather insensitive to temperature, but becomes more rapid at high ionic strength or at basic pH. In a 0.25 M ionic strength buffer, the half-life of the complex is about 1 .O min. The rate of association is very fast and follows secondorder kinetics with the rate constant for association (ka) being 8 x lo7 M-' sC1 at 30 "C in the standard buffer. The rate of association is almost insensitive to ionic strength but slightly sensitive to pH or temperature. Monovalent cations can also promote the binding reaction as well as divalent cations but the complex formed with monovalent cation is unstable. A study of the kinetics of dissociation suggests that there are two types of interaction between MS2 phage and F pilus; one is a strong interaction formed with divalent cations and the other is a weak one formed with monovalent cations. The physical nature of the bonds involved in the former and the latter seems to be mainly electrostatic and non-electrostatic respectively. The mechanism of the binding reaction is discussed.Various types of molecules accessible from the bacterial envelope (protein, lipopolysaccharide, capsular polysaccharide, etc.) and appendages on the cell surface (flagella, sex pili) can serve as phage receptors. Adsorption of male-specific coliphage is attributed to F pilus [l -41, a class of sex pili which are a phenotypic expression of the fertility F factor [2]. Spherical RNA phages adsorb to F pilus in large numbers along the entire length of F pilus [1,2], while filamentous DNA phages adsorb to the tip of the pilus in limited numbers [3]. The binding of the RNA phage to the side of F pilus appears to be facilitated by the A protein component of the phage [5,6], and the isolation of a number of F pilus bacterial mutants with altered phage attachment properties [7-91 indicates that the adsorption process involves a highly specific protein -protein interactionThe interaction between the RNA phage and free F pilus has been studied by retention of the phagepilus complex on nitrocellulose filters [4,11,12]. There [lo].Ahhreviafions. kdr rate constant of dissociation; k,, rate constant of association; K , equilibrium constant ( k d / k a ) ; f l i 2 , dissociation half-life M-F complex, MS2-phage-F-pilus complex.Dqfinifion. Standard buffer, 10 mM Tris-HCI, pH 7.2, 5 mM CaCI2, and 20 pg/ml of streptomycin sulfate.is no specific requirement for the reaction and there is only a slight temperature dependence, the binding at 0 "C being about half that 37 "C 14,111. The binding reaction of the phage to F pilus is a reversible one [4], having an association constant of 1.75 at 4 "C when cell-bound F pili are used [13]. Very little is known, however, about t...

Analysis of Hydroxyl Radicals and Inactivation Mechanisms of Bacteriophage MS2 in Response to a Simultaneous Application of UV and Chlorine

“…E. coli K12 A/λ F + was incubated in Lennox L broth base (LB broth) (Invitrogen) at 37 °C for 2.5 h and then centrifuged at 6,000 rpm for 10 min. The supernatant was removed and the pellet was resuspended with 0.07 mM PBS, in which phage attachment without phage development is allowed ,, (SI Figure S6), to obtain an optical density (OD 600 ) of 0.2 cm –1 (∼1.6 × 10 6 CFU/mL). The E. coli suspension was mixed with equal volumes of the control (untreated MS2) or disinfected samples (treated MS2), with a multiplicity of infection (MOI, a ratio between number of viruses and host cells and details can be found in SI, Text S3) of 0.01 for both control and disinfected samples .…”

The Phage Infection Process: a Functional Role for the Distal Linker Region of Bacteriophage Protein 3

“…Since the N1L1 phage, in our experiments, seemed to be dependent on the presence of the F pilus, it was important to elucidate if the engineered phage physically interacted with the primary receptor ( Table 1). The fact that an extracellular level of divalent cations was required for infection with RNA phage (8,22) was used to set up an assay where the F pilus was blocked by the RNA phage MS2. Thus, no translocation of MS2 RNA took place in the absence of cations.…”

Structural basis for the adsorption of a single-stranded RNA bacteriophage

“…All known varieties of retractile pili in Proteobacteria can serve as the receptor of ssRNA phages, including conjugative pili (e.g. F and M) 6,8,9 , type IV twitching motility pili 10 , and species-specific pili, such as the Tad pili of swarmer Caulobacter cells 11 . It should be noted that ssRNA phages specific for the pili encoded on conjugal plasmids may not have a required bacterial host as these plasmids can have a wide host range 12 .…”