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Development of a novel system for isolating genes involved in predator-prey interactions using host independent derivatives of Bdellovibrio bacteriovorus 109J.

Medina AA, Shanks RM, Kadouri DE - BMC Microbiol. (2008)

Bottom Line: Ten HI transposon mutants mapped to genes predicted to be involved in mechanisms previously implicated in predation (flagella, pili and chemotaxis) were further examined for their ability to reduce biofilms.Furthermore, genes identified in this study suggest that surface gliding motility may also play a role in predation of biofilms consistent with Bdellovibrios occupying a biofilm niche.We believe that the methodology presented here will open the way for future studies on the mechanisms involved in Bdellovibrio host-prey interaction and a greater insight of the biology of this unique organism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07101, USA. medinaaa@umdnj.edu

ABSTRACT

Background: Bdellovibrio bacteriovorus is a gram-negative bacterium that preys upon other gram-negative bacteria. Although the life cycle of Bdellovibrio has been extensively investigated, very little is known about the mechanisms involved in predation.

Results: Host-Independent (HI) mutants of B. bacteriovorus were isolated from wild-type strain 109J. Predation assays confirmed that the selected HI mutants retained their ability to prey on host cells grown planktonically and in a biofilm. A mariner transposon library of B. bacteriovorus HI was constructed and HI mutants that were impaired in their ability to attack biofilms were isolated. Transposon insertion sites were determined using arbitrary polymerase chain reaction. Ten HI transposon mutants mapped to genes predicted to be involved in mechanisms previously implicated in predation (flagella, pili and chemotaxis) were further examined for their ability to reduce biofilms.

Conclusion: In this study we describe a new method for isolating genes that are required for Bdellovibrio biofilm predation. Focusing on mechanisms that were previously attributed to be involved in predation, we demonstrate that motility systems are required for predation of bacterial biofilms. Furthermore, genes identified in this study suggest that surface gliding motility may also play a role in predation of biofilms consistent with Bdellovibrios occupying a biofilm niche. We believe that the methodology presented here will open the way for future studies on the mechanisms involved in Bdellovibrio host-prey interaction and a greater insight of the biology of this unique organism.

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Biofilm predation by HI biofilm predation mutants (BPM). Ten HI biofilm predation mutants (BPM-5, 14, 15, 6, 7, 13, 20, 28, 37, 8), HI transposon insertion mutants (HI-Ra, HI-Rb) and HI mutant A (HI-A) were grown and used for the following assays: (A) Plaque predation assays. The above samples were spotted on a thick lawn of host cells (pre-treatment). Images were taken 48 hr post-inoculation. Each experiment was carried out three times, with three replicates for each treatment, yielding similar results- representative images are shown here. (B) Biofilm predation assays. E. coli biofilms were developed for 18 hrs in 96 well microtiter plates (pre-treatment), followed by 48 hr exposure to various treatments then rinsed and stained with CV. Each experiment was carried out three times, with 24 wells for each treatment, yielding similar results. (C) Quantification of biofilm biomass. Samples were added to a developed E. coli biofilm. Forty-eight hours later the dishes were rinsed, stained with CV and the amount of CV staining was quantified at OD600 for each time point. Each value represents the mean of 12 wells from one representative experiment. Error bars indicate standard errors. Each experiment was carried out three times yielding similar results. The difference in biofilm reduction between the biofilm reducing mutants (BPM-5, 14, 15, 6, 7, 13, 20, 28, 37, 8), DDNB control, and the treatments which were able to reduce the pre-developed biofilm (HI-A, HI-Ra, HI-Rb) was statistically significant (P < 0.001).
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Figure 3: Biofilm predation by HI biofilm predation mutants (BPM). Ten HI biofilm predation mutants (BPM-5, 14, 15, 6, 7, 13, 20, 28, 37, 8), HI transposon insertion mutants (HI-Ra, HI-Rb) and HI mutant A (HI-A) were grown and used for the following assays: (A) Plaque predation assays. The above samples were spotted on a thick lawn of host cells (pre-treatment). Images were taken 48 hr post-inoculation. Each experiment was carried out three times, with three replicates for each treatment, yielding similar results- representative images are shown here. (B) Biofilm predation assays. E. coli biofilms were developed for 18 hrs in 96 well microtiter plates (pre-treatment), followed by 48 hr exposure to various treatments then rinsed and stained with CV. Each experiment was carried out three times, with 24 wells for each treatment, yielding similar results. (C) Quantification of biofilm biomass. Samples were added to a developed E. coli biofilm. Forty-eight hours later the dishes were rinsed, stained with CV and the amount of CV staining was quantified at OD600 for each time point. Each value represents the mean of 12 wells from one representative experiment. Error bars indicate standard errors. Each experiment was carried out three times yielding similar results. The difference in biofilm reduction between the biofilm reducing mutants (BPM-5, 14, 15, 6, 7, 13, 20, 28, 37, 8), DDNB control, and the treatments which were able to reduce the pre-developed biofilm (HI-A, HI-Ra, HI-Rb) was statistically significant (P < 0.001).

Mentions: It was previously demonstrated that swimming motility is required for the predatory lifecycle of B. bacteriovorus [9,11]. We have identified the B. bacteriovorus 109J homologues of two flgE genes and a flgJ gene, based on the degree of similarity of the predicted polypeptide encoded by the DNA sequence flanking the insertion in the strain carrying allele BPM-5, 14 and 15 to the B. bacteriovorus HD 100 flgE genes and flgJ (Table 1). Both flgE and flgJ are thought to participate in flagellar hook and rod assembly [20,21]. When spotted on a lawn of host bacteria, BPM-5, 14 and 15 were unable to form lytic halos (Fig. 3A, BPM-5, 14, 15). Furthermore no reduction of E. coli biofilm was detected following a 48 hr incubation period with the selected mutants (Fig. 3B, BPM-5, 14, 15). A reduction of 7.7%, 14% and 16.4% in CV staining was measured following a 48 hr incubation period with BPM-5, 14 and 15 respectively, compared to 70% decrease in the biofilms treated with the recipient HI-A (Fig. 3B–C).


Development of a novel system for isolating genes involved in predator-prey interactions using host independent derivatives of Bdellovibrio bacteriovorus 109J.

Medina AA, Shanks RM, Kadouri DE - BMC Microbiol. (2008)

Biofilm predation by HI biofilm predation mutants (BPM). Ten HI biofilm predation mutants (BPM-5, 14, 15, 6, 7, 13, 20, 28, 37, 8), HI transposon insertion mutants (HI-Ra, HI-Rb) and HI mutant A (HI-A) were grown and used for the following assays: (A) Plaque predation assays. The above samples were spotted on a thick lawn of host cells (pre-treatment). Images were taken 48 hr post-inoculation. Each experiment was carried out three times, with three replicates for each treatment, yielding similar results- representative images are shown here. (B) Biofilm predation assays. E. coli biofilms were developed for 18 hrs in 96 well microtiter plates (pre-treatment), followed by 48 hr exposure to various treatments then rinsed and stained with CV. Each experiment was carried out three times, with 24 wells for each treatment, yielding similar results. (C) Quantification of biofilm biomass. Samples were added to a developed E. coli biofilm. Forty-eight hours later the dishes were rinsed, stained with CV and the amount of CV staining was quantified at OD600 for each time point. Each value represents the mean of 12 wells from one representative experiment. Error bars indicate standard errors. Each experiment was carried out three times yielding similar results. The difference in biofilm reduction between the biofilm reducing mutants (BPM-5, 14, 15, 6, 7, 13, 20, 28, 37, 8), DDNB control, and the treatments which were able to reduce the pre-developed biofilm (HI-A, HI-Ra, HI-Rb) was statistically significant (P < 0.001).
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Figure 3: Biofilm predation by HI biofilm predation mutants (BPM). Ten HI biofilm predation mutants (BPM-5, 14, 15, 6, 7, 13, 20, 28, 37, 8), HI transposon insertion mutants (HI-Ra, HI-Rb) and HI mutant A (HI-A) were grown and used for the following assays: (A) Plaque predation assays. The above samples were spotted on a thick lawn of host cells (pre-treatment). Images were taken 48 hr post-inoculation. Each experiment was carried out three times, with three replicates for each treatment, yielding similar results- representative images are shown here. (B) Biofilm predation assays. E. coli biofilms were developed for 18 hrs in 96 well microtiter plates (pre-treatment), followed by 48 hr exposure to various treatments then rinsed and stained with CV. Each experiment was carried out three times, with 24 wells for each treatment, yielding similar results. (C) Quantification of biofilm biomass. Samples were added to a developed E. coli biofilm. Forty-eight hours later the dishes were rinsed, stained with CV and the amount of CV staining was quantified at OD600 for each time point. Each value represents the mean of 12 wells from one representative experiment. Error bars indicate standard errors. Each experiment was carried out three times yielding similar results. The difference in biofilm reduction between the biofilm reducing mutants (BPM-5, 14, 15, 6, 7, 13, 20, 28, 37, 8), DDNB control, and the treatments which were able to reduce the pre-developed biofilm (HI-A, HI-Ra, HI-Rb) was statistically significant (P < 0.001).
Mentions: It was previously demonstrated that swimming motility is required for the predatory lifecycle of B. bacteriovorus [9,11]. We have identified the B. bacteriovorus 109J homologues of two flgE genes and a flgJ gene, based on the degree of similarity of the predicted polypeptide encoded by the DNA sequence flanking the insertion in the strain carrying allele BPM-5, 14 and 15 to the B. bacteriovorus HD 100 flgE genes and flgJ (Table 1). Both flgE and flgJ are thought to participate in flagellar hook and rod assembly [20,21]. When spotted on a lawn of host bacteria, BPM-5, 14 and 15 were unable to form lytic halos (Fig. 3A, BPM-5, 14, 15). Furthermore no reduction of E. coli biofilm was detected following a 48 hr incubation period with the selected mutants (Fig. 3B, BPM-5, 14, 15). A reduction of 7.7%, 14% and 16.4% in CV staining was measured following a 48 hr incubation period with BPM-5, 14 and 15 respectively, compared to 70% decrease in the biofilms treated with the recipient HI-A (Fig. 3B–C).

Bottom Line: Ten HI transposon mutants mapped to genes predicted to be involved in mechanisms previously implicated in predation (flagella, pili and chemotaxis) were further examined for their ability to reduce biofilms.Furthermore, genes identified in this study suggest that surface gliding motility may also play a role in predation of biofilms consistent with Bdellovibrios occupying a biofilm niche.We believe that the methodology presented here will open the way for future studies on the mechanisms involved in Bdellovibrio host-prey interaction and a greater insight of the biology of this unique organism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07101, USA. medinaaa@umdnj.edu

ABSTRACT

Background: Bdellovibrio bacteriovorus is a gram-negative bacterium that preys upon other gram-negative bacteria. Although the life cycle of Bdellovibrio has been extensively investigated, very little is known about the mechanisms involved in predation.

Results: Host-Independent (HI) mutants of B. bacteriovorus were isolated from wild-type strain 109J. Predation assays confirmed that the selected HI mutants retained their ability to prey on host cells grown planktonically and in a biofilm. A mariner transposon library of B. bacteriovorus HI was constructed and HI mutants that were impaired in their ability to attack biofilms were isolated. Transposon insertion sites were determined using arbitrary polymerase chain reaction. Ten HI transposon mutants mapped to genes predicted to be involved in mechanisms previously implicated in predation (flagella, pili and chemotaxis) were further examined for their ability to reduce biofilms.

Conclusion: In this study we describe a new method for isolating genes that are required for Bdellovibrio biofilm predation. Focusing on mechanisms that were previously attributed to be involved in predation, we demonstrate that motility systems are required for predation of bacterial biofilms. Furthermore, genes identified in this study suggest that surface gliding motility may also play a role in predation of biofilms consistent with Bdellovibrios occupying a biofilm niche. We believe that the methodology presented here will open the way for future studies on the mechanisms involved in Bdellovibrio host-prey interaction and a greater insight of the biology of this unique organism.

Show MeSH
Related in: MedlinePlus