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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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Transconjugant fraction over time with varying levels of phage or phagemid particles.For visual clarity, each line represents a single experiment, although multiple experiments were performed to accurately determine the conjugation rate. Inhibition of conjugation by replicating M13 phage: (A) M13-kmR (dark blue = not infected; for pre-infected F+ cells, light blue = 107 pfu/mL, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL); (B) M13-ampR (dark blue = not infected; for pre-infected F+ cells, light blue = 106pfu/mL, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL); (C) M13-kmR (F+ cells were not pre-infected; blue = not infected, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL). (D) Inhibition of conjugation by non-replicating phagemid particles lacking phage genes: phagemid particles (blue = no phagemid particles added, green = 108 cfu/mL, yellow = 109 cfu/mL, orange = 1010 cfu/mL, red = 1011 cfu/mL).
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pone-0019991-g002: Transconjugant fraction over time with varying levels of phage or phagemid particles.For visual clarity, each line represents a single experiment, although multiple experiments were performed to accurately determine the conjugation rate. Inhibition of conjugation by replicating M13 phage: (A) M13-kmR (dark blue = not infected; for pre-infected F+ cells, light blue = 107 pfu/mL, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL); (B) M13-ampR (dark blue = not infected; for pre-infected F+ cells, light blue = 106pfu/mL, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL); (C) M13-kmR (F+ cells were not pre-infected; blue = not infected, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL). (D) Inhibition of conjugation by non-replicating phagemid particles lacking phage genes: phagemid particles (blue = no phagemid particles added, green = 108 cfu/mL, yellow = 109 cfu/mL, orange = 1010 cfu/mL, red = 1011 cfu/mL).

Mentions: We modified the commercially available phage M13KE by inserting a kanamycin resistance cassette into the multiple cloning site, thus enabling selection for infected cells when desired. As expected, cells infected with M13-kmR exhibited a slower growth rate (Table 1). To verify that M13-kmR inhibits conjugation, we grew a culture of F+ cells infected by M13-kmR under selection for kanamycin resistance to prevent loss of infection (donor cells). We further added varying amounts of purified M13-kmR phage to the media at the beginning of the conjugation experiment. We found that infected F+ cells conjugated with slightly reduced but still good efficiency at low levels of externally added phage (e.g., 107 particles/mL), but the conjugation rate decreased as the phage concentration increased, such that almost no transconjugants were detected if the concentration of phage was ≥1011 particles/mL (Figure 2A). To check that this effect was not dependent on the specific plasmids carrying fluorescent markers or on kanamycin resistance, we also performed these experiments with F+ and F− cells carrying marker plasmids based on pRG5, with the F+ cells infected by M13-ampR. Similar results were obtained in this alternative system (Figure 2B).


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)

Transconjugant fraction over time with varying levels of phage or phagemid particles.For visual clarity, each line represents a single experiment, although multiple experiments were performed to accurately determine the conjugation rate. Inhibition of conjugation by replicating M13 phage: (A) M13-kmR (dark blue = not infected; for pre-infected F+ cells, light blue = 107 pfu/mL, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL); (B) M13-ampR (dark blue = not infected; for pre-infected F+ cells, light blue = 106pfu/mL, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL); (C) M13-kmR (F+ cells were not pre-infected; blue = not infected, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL). (D) Inhibition of conjugation by non-replicating phagemid particles lacking phage genes: phagemid particles (blue = no phagemid particles added, green = 108 cfu/mL, yellow = 109 cfu/mL, orange = 1010 cfu/mL, red = 1011 cfu/mL).
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pone-0019991-g002: Transconjugant fraction over time with varying levels of phage or phagemid particles.For visual clarity, each line represents a single experiment, although multiple experiments were performed to accurately determine the conjugation rate. Inhibition of conjugation by replicating M13 phage: (A) M13-kmR (dark blue = not infected; for pre-infected F+ cells, light blue = 107 pfu/mL, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL); (B) M13-ampR (dark blue = not infected; for pre-infected F+ cells, light blue = 106pfu/mL, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL); (C) M13-kmR (F+ cells were not pre-infected; blue = not infected, green = 108 pfu/mL, yellow = 109 pfu/mL, orange = 1010 pfu/mL, red = 1011 pfu/mL). (D) Inhibition of conjugation by non-replicating phagemid particles lacking phage genes: phagemid particles (blue = no phagemid particles added, green = 108 cfu/mL, yellow = 109 cfu/mL, orange = 1010 cfu/mL, red = 1011 cfu/mL).
Mentions: We modified the commercially available phage M13KE by inserting a kanamycin resistance cassette into the multiple cloning site, thus enabling selection for infected cells when desired. As expected, cells infected with M13-kmR exhibited a slower growth rate (Table 1). To verify that M13-kmR inhibits conjugation, we grew a culture of F+ cells infected by M13-kmR under selection for kanamycin resistance to prevent loss of infection (donor cells). We further added varying amounts of purified M13-kmR phage to the media at the beginning of the conjugation experiment. We found that infected F+ cells conjugated with slightly reduced but still good efficiency at low levels of externally added phage (e.g., 107 particles/mL), but the conjugation rate decreased as the phage concentration increased, such that almost no transconjugants were detected if the concentration of phage was ≥1011 particles/mL (Figure 2A). To check that this effect was not dependent on the specific plasmids carrying fluorescent markers or on kanamycin resistance, we also performed these experiments with F+ and F− cells carrying marker plasmids based on pRG5, with the F+ cells infected by M13-ampR. Similar results were obtained in this alternative system (Figure 2B).

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