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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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Model and experimental data for phage replication in F+ culture (all cells infected).(A) Replication rate of phage. The y-axis is the phage (red) or cell (blue) concentration over time. Experimental data are shown with the dotted lines; model fit is shown with the solid lines (kr = 60 /hr, RMSD = 3×109 units/mL). (B) Model of inhibition of conjugation by replicating phage: Example of conjugation data (in this example, M13-kmR was added to 109 pfu/mL; cells were pre-infected), with fit to determine R and ki. Data are shown in red and purple (two replicates), with model fit shown in blue (R = 0.2, ki = 10−8 /hr; RMSD over all conjugation trials = 0.32).
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pone-0019991-g006: Model and experimental data for phage replication in F+ culture (all cells infected).(A) Replication rate of phage. The y-axis is the phage (red) or cell (blue) concentration over time. Experimental data are shown with the dotted lines; model fit is shown with the solid lines (kr = 60 /hr, RMSD = 3×109 units/mL). (B) Model of inhibition of conjugation by replicating phage: Example of conjugation data (in this example, M13-kmR was added to 109 pfu/mL; cells were pre-infected), with fit to determine R and ki. Data are shown in red and purple (two replicates), with model fit shown in blue (R = 0.2, ki = 10−8 /hr; RMSD over all conjugation trials = 0.32).

Mentions: The conjugative ability of infected cells was qualitatively lower than that of non-infected cells (Figure 2A,C), especially at low phage concentrations. To quantify this difference, we first determined the replication rate of phage in order to model the phage concentration in the media throughout the experiment. We then fit the cell and phage concentration over time to a model of growth and replication, described by the following equations:We determined kginfected from independent cell density measurements in liquid culture. Therefore, we fit the phage replication data with a single parameter kr to obtain kr = 60 /hr (Table 1; Figure 6A), i.e., about 60 phage particles are produced per hour by each infected cell.


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)

Model and experimental data for phage replication in F+ culture (all cells infected).(A) Replication rate of phage. The y-axis is the phage (red) or cell (blue) concentration over time. Experimental data are shown with the dotted lines; model fit is shown with the solid lines (kr = 60 /hr, RMSD = 3×109 units/mL). (B) Model of inhibition of conjugation by replicating phage: Example of conjugation data (in this example, M13-kmR was added to 109 pfu/mL; cells were pre-infected), with fit to determine R and ki. Data are shown in red and purple (two replicates), with model fit shown in blue (R = 0.2, ki = 10−8 /hr; RMSD over all conjugation trials = 0.32).
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Related In: Results  -  Collection

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

pone-0019991-g006: Model and experimental data for phage replication in F+ culture (all cells infected).(A) Replication rate of phage. The y-axis is the phage (red) or cell (blue) concentration over time. Experimental data are shown with the dotted lines; model fit is shown with the solid lines (kr = 60 /hr, RMSD = 3×109 units/mL). (B) Model of inhibition of conjugation by replicating phage: Example of conjugation data (in this example, M13-kmR was added to 109 pfu/mL; cells were pre-infected), with fit to determine R and ki. Data are shown in red and purple (two replicates), with model fit shown in blue (R = 0.2, ki = 10−8 /hr; RMSD over all conjugation trials = 0.32).
Mentions: The conjugative ability of infected cells was qualitatively lower than that of non-infected cells (Figure 2A,C), especially at low phage concentrations. To quantify this difference, we first determined the replication rate of phage in order to model the phage concentration in the media throughout the experiment. We then fit the cell and phage concentration over time to a model of growth and replication, described by the following equations:We determined kginfected from independent cell density measurements in liquid culture. Therefore, we fit the phage replication data with a single parameter kr to obtain kr = 60 /hr (Table 1; Figure 6A), i.e., about 60 phage particles are produced per hour by each infected cell.

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