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Inhibition of bacterial conjugation by phage M13 and its protein g3p: quantitative analysis and model.

Lin A, Jimenez J, Derr J, Vera P, Manapat ML, Esvelt KM, Villanueva L, Liu DR, Chen IA - PLoS ONE (2011)

Bottom Line: Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances.We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration.Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

View Article: PubMed Central - PubMed

Affiliation: FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, United States of America.

ABSTRACT
Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

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Inhibition of conjugation by non-replicating phagemid particles lacking phage genes: Inhibition curve for conjugation reflects the association between phagemid particles and F+ cells.The y-axis is the conjugation rate k normalized by the conjugation rate in the absence of phage (k0); error bars are standard deviation from replicates. The line represents the fit to a binding equilibrium (Kd = 2 pM; RMSD = 0.03).
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pone-0019991-g003: Inhibition of conjugation by non-replicating phagemid particles lacking phage genes: Inhibition curve for conjugation reflects the association between phagemid particles and F+ cells.The y-axis is the conjugation rate k normalized by the conjugation rate in the absence of phage (k0); error bars are standard deviation from replicates. The line represents the fit to a binding equilibrium (Kd = 2 pM; RMSD = 0.03).

Mentions: We hypothesized that the phagemid particles inhibit conjugation by simply binding to the tip of the F pilus and rendering them incapable of contacting potential recipient cells. We modeled this as a binding equilibrium between F+ cells and phagemid particles, with dissociation constant Kd, in which the complex formed is incapable of conjugation (i.e., [phage][F+] = Kd [complex]). This effectively reduces the concentration of competent donor F+ cells, such that the ratio α of the conjugation rate in the presence of phage particles (kcphage) to the basal conjugation rate (kc) is given by α = [F+]/([F+]+[complex]) = 1/(1+[phage]/Kd). We fit the conjugation data in the presence of the phagemid particles to the simple model of conjugation and determined kcphage for several phage concentrations. The relationship between relative conjugation rate and phage concentration could be fit using the binding equilibrium (Figure 3), with apparent Kd = 2 pM. This model could also be naively applied to conjugation kinetics in the presence of replicating phage (apparent Kd = 2.4 pM; Figure S1). However, the concentration of phage increases over time due to infection and replication, so this procedure is likely to underestimate the true affinity of the interaction.


Inhibition of bacterial conjugation by phage M13 and its protein g3p: quantitative analysis and model.

Lin A, Jimenez J, Derr J, Vera P, Manapat ML, Esvelt KM, Villanueva L, Liu DR, Chen IA - PLoS ONE (2011)

Inhibition of conjugation by non-replicating phagemid particles lacking phage genes: Inhibition curve for conjugation reflects the association between phagemid particles and F+ cells.The y-axis is the conjugation rate k normalized by the conjugation rate in the absence of phage (k0); error bars are standard deviation from replicates. The line represents the fit to a binding equilibrium (Kd = 2 pM; RMSD = 0.03).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102678&req=5

pone-0019991-g003: Inhibition of conjugation by non-replicating phagemid particles lacking phage genes: Inhibition curve for conjugation reflects the association between phagemid particles and F+ cells.The y-axis is the conjugation rate k normalized by the conjugation rate in the absence of phage (k0); error bars are standard deviation from replicates. The line represents the fit to a binding equilibrium (Kd = 2 pM; RMSD = 0.03).
Mentions: We hypothesized that the phagemid particles inhibit conjugation by simply binding to the tip of the F pilus and rendering them incapable of contacting potential recipient cells. We modeled this as a binding equilibrium between F+ cells and phagemid particles, with dissociation constant Kd, in which the complex formed is incapable of conjugation (i.e., [phage][F+] = Kd [complex]). This effectively reduces the concentration of competent donor F+ cells, such that the ratio α of the conjugation rate in the presence of phage particles (kcphage) to the basal conjugation rate (kc) is given by α = [F+]/([F+]+[complex]) = 1/(1+[phage]/Kd). We fit the conjugation data in the presence of the phagemid particles to the simple model of conjugation and determined kcphage for several phage concentrations. The relationship between relative conjugation rate and phage concentration could be fit using the binding equilibrium (Figure 3), with apparent Kd = 2 pM. This model could also be naively applied to conjugation kinetics in the presence of replicating phage (apparent Kd = 2.4 pM; Figure S1). However, the concentration of phage increases over time due to infection and replication, so this procedure is likely to underestimate the true affinity of the interaction.

Bottom Line: Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances.We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration.Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

View Article: PubMed Central - PubMed

Affiliation: FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, United States of America.

ABSTRACT
Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

Show MeSH
Related in: MedlinePlus