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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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Predation by B. bacteriovorus wt and HI mutants. (A) Plaque predation assays. Wild-type B. bacteriovorus lysates (B.b wt) or HI mutants (HI-A, B, C) were grown and transferred to a thick lawn of K. pneumoniae host cells (pre-treatment). DDNB and heat killed (30 min at 90°C) HI mutant A were used as negative controls. Forty-eight hours after inoculation a clear lytic halo formed at the point of 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 B. bacteriovorus lysate, HI mutants (HI-A, B, C), DDNB or heat killed HI mutant A, then rinsed and stained with CV. Each experiment was carried out three times, with 24 wells for each treatment, yielding similar results- representative images are shown here. (C) Quantification of biofilm biomass. B. bacteriovorus lysate, HI mutants, DDNB or heat killed HI mutant A, 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 B. bacteriovorus lysate, HI-A, B, C and the negative controls (DDNB and the heat killed HI-A) was statistically significant (P < 0.001). (D) Cell viability counts of planktonic E. coli. Planktonic E. coli cells were mixed with B. bacteriovorus lysate, HI mutants (HI-A, B, C), DDNB or heat killed HI mutant A, and the bacterial viability counts determined. Each experiment was carried out three times yielding similar results. Each value represents the mean of 3 lysates from one representative experiment. Error bars indicate standard errors. The difference in host viability at 24 hr between B. bacteriovorus lysate, HI-A, B, C and the negative controls (DDNB and the heat killed HI-A) was statistically significant (P < 0.001). The difference in host viability at 24 hr between B. bacteriovorus lysate and HI-A was statistically significant (P = 0.05).
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Figure 1: Predation by B. bacteriovorus wt and HI mutants. (A) Plaque predation assays. Wild-type B. bacteriovorus lysates (B.b wt) or HI mutants (HI-A, B, C) were grown and transferred to a thick lawn of K. pneumoniae host cells (pre-treatment). DDNB and heat killed (30 min at 90°C) HI mutant A were used as negative controls. Forty-eight hours after inoculation a clear lytic halo formed at the point of 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 B. bacteriovorus lysate, HI mutants (HI-A, B, C), DDNB or heat killed HI mutant A, then rinsed and stained with CV. Each experiment was carried out three times, with 24 wells for each treatment, yielding similar results- representative images are shown here. (C) Quantification of biofilm biomass. B. bacteriovorus lysate, HI mutants, DDNB or heat killed HI mutant A, 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 B. bacteriovorus lysate, HI-A, B, C and the negative controls (DDNB and the heat killed HI-A) was statistically significant (P < 0.001). (D) Cell viability counts of planktonic E. coli. Planktonic E. coli cells were mixed with B. bacteriovorus lysate, HI mutants (HI-A, B, C), DDNB or heat killed HI mutant A, and the bacterial viability counts determined. Each experiment was carried out three times yielding similar results. Each value represents the mean of 3 lysates from one representative experiment. Error bars indicate standard errors. The difference in host viability at 24 hr between B. bacteriovorus lysate, HI-A, B, C and the negative controls (DDNB and the heat killed HI-A) was statistically significant (P < 0.001). The difference in host viability at 24 hr between B. bacteriovorus lysate and HI-A was statistically significant (P = 0.05).

Mentions: Using an HI enrichment protocol [18] twenty-five HI mutants were isolated from six independent enrichment cultures. The selected HI colonies were evaluated by PCR, using 16S rRNA primers that specifically target Bdellovibrionaceae [19] and primers that amplify the hit locus of B. bacteriovorus [16]. PCR reactions had confirmed that the selected HI colonies were derivatives of Bdellovibrio (data not shown). Sequence analysis of the hit locus revealed no sequence deviation between B. bacteriovorus 109J WT strain and the HI-A variant, as was previously noted for other HI variants [16]. In order to assess the facultative behavior of the HI mutants and to demonstrate that the mutants retained their ability to attack surface attached and planktonicly grown host cells, three random HI mutants (HI-A, B, C) were spotted on a lawn of host bacteria. After 48 hr, a clear lytic halo appeared at the point of inoculation (Fig, 1A, HI-A, B, C). A lytic halo also appeared where the filtered B. bacteriovorus wild-type lysate (contains B. bacteriovorus) was spotted (Fig, 1A, B.b WT) but did not emerge where DDNB buffer alone (Fig, 1A, DDNB) or heat killed HI-A mutant were inoculated (Fig, 1A, Heat Killed HI-A). Additionally, the effects of B. bacteriovorus HI mutants on E. coli biofilms were measured. E. coli biofilms (comprised of ~1 × 108 cfu/well) were formed in 96 well microtiter plates for ~18 hrs. Thereafter the medium was removed and the wells were washed with DDNB medium as described in the Materials and Methods. The E. coli biofilms were exposed for 48 hr to the HI mutants, B. bacteriovorus lysate or DDNB. As shown in Fig. 1B (pre-treatment), the untreated 18 hr-old biofilm was easily visualized with CV-staining. Treatment with 1 × 107 pfu of B. bacteriovorus (Fig 1B, B.b WT) or 1 × 107 cfu HI mutants (Fig. 1B, HI-A, B, C) markedly reduced the CV-staining compared to the DDNB or heat killed HI-A control (Fig. 1B, DDNB, and Heat Killed HI-A). Quantification of the effect of B. bacteriovorus on E. coli biofilms over time revealed a 69% reduction in CV staining at 24 hr post-treatment and an 81% reduction after 48 hr (Fig. 1C, B.b WT), compared to the initial time point (pre-treatment). A reduction of 63%, 55%, and 52% was observed following a 24 hr exposure period to HI mutants A, B, C, and a decrease of 70%, 62%, and 63% following 48 hr of incubation (Fig. 1C, HI-A, B, C). In contrast, only a 22% and 16.4% reduction in CV staining was measured after 48 hr in the control sample treated with DDNB and heat killed HI-A respectively (Fig. 1C, DDNB, and Heat Killed HI-A). The ability of the HI mutants to reduce host cells grown planktonicly was also examined in standard induced lysates. All HI mutants, as well as B. bacteriovorus were able to reduce the planktonic population by ~5 logs in the first 24 hr of predation with no reduction occurring when DDNB alone or HI heat killed mutant A was added (Fig. 1D).


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)

Predation by B. bacteriovorus wt and HI mutants. (A) Plaque predation assays. Wild-type B. bacteriovorus lysates (B.b wt) or HI mutants (HI-A, B, C) were grown and transferred to a thick lawn of K. pneumoniae host cells (pre-treatment). DDNB and heat killed (30 min at 90°C) HI mutant A were used as negative controls. Forty-eight hours after inoculation a clear lytic halo formed at the point of 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 B. bacteriovorus lysate, HI mutants (HI-A, B, C), DDNB or heat killed HI mutant A, then rinsed and stained with CV. Each experiment was carried out three times, with 24 wells for each treatment, yielding similar results- representative images are shown here. (C) Quantification of biofilm biomass. B. bacteriovorus lysate, HI mutants, DDNB or heat killed HI mutant A, 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 B. bacteriovorus lysate, HI-A, B, C and the negative controls (DDNB and the heat killed HI-A) was statistically significant (P < 0.001). (D) Cell viability counts of planktonic E. coli. Planktonic E. coli cells were mixed with B. bacteriovorus lysate, HI mutants (HI-A, B, C), DDNB or heat killed HI mutant A, and the bacterial viability counts determined. Each experiment was carried out three times yielding similar results. Each value represents the mean of 3 lysates from one representative experiment. Error bars indicate standard errors. The difference in host viability at 24 hr between B. bacteriovorus lysate, HI-A, B, C and the negative controls (DDNB and the heat killed HI-A) was statistically significant (P < 0.001). The difference in host viability at 24 hr between B. bacteriovorus lysate and HI-A was statistically significant (P = 0.05).
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Figure 1: Predation by B. bacteriovorus wt and HI mutants. (A) Plaque predation assays. Wild-type B. bacteriovorus lysates (B.b wt) or HI mutants (HI-A, B, C) were grown and transferred to a thick lawn of K. pneumoniae host cells (pre-treatment). DDNB and heat killed (30 min at 90°C) HI mutant A were used as negative controls. Forty-eight hours after inoculation a clear lytic halo formed at the point of 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 B. bacteriovorus lysate, HI mutants (HI-A, B, C), DDNB or heat killed HI mutant A, then rinsed and stained with CV. Each experiment was carried out three times, with 24 wells for each treatment, yielding similar results- representative images are shown here. (C) Quantification of biofilm biomass. B. bacteriovorus lysate, HI mutants, DDNB or heat killed HI mutant A, 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 B. bacteriovorus lysate, HI-A, B, C and the negative controls (DDNB and the heat killed HI-A) was statistically significant (P < 0.001). (D) Cell viability counts of planktonic E. coli. Planktonic E. coli cells were mixed with B. bacteriovorus lysate, HI mutants (HI-A, B, C), DDNB or heat killed HI mutant A, and the bacterial viability counts determined. Each experiment was carried out three times yielding similar results. Each value represents the mean of 3 lysates from one representative experiment. Error bars indicate standard errors. The difference in host viability at 24 hr between B. bacteriovorus lysate, HI-A, B, C and the negative controls (DDNB and the heat killed HI-A) was statistically significant (P < 0.001). The difference in host viability at 24 hr between B. bacteriovorus lysate and HI-A was statistically significant (P = 0.05).
Mentions: Using an HI enrichment protocol [18] twenty-five HI mutants were isolated from six independent enrichment cultures. The selected HI colonies were evaluated by PCR, using 16S rRNA primers that specifically target Bdellovibrionaceae [19] and primers that amplify the hit locus of B. bacteriovorus [16]. PCR reactions had confirmed that the selected HI colonies were derivatives of Bdellovibrio (data not shown). Sequence analysis of the hit locus revealed no sequence deviation between B. bacteriovorus 109J WT strain and the HI-A variant, as was previously noted for other HI variants [16]. In order to assess the facultative behavior of the HI mutants and to demonstrate that the mutants retained their ability to attack surface attached and planktonicly grown host cells, three random HI mutants (HI-A, B, C) were spotted on a lawn of host bacteria. After 48 hr, a clear lytic halo appeared at the point of inoculation (Fig, 1A, HI-A, B, C). A lytic halo also appeared where the filtered B. bacteriovorus wild-type lysate (contains B. bacteriovorus) was spotted (Fig, 1A, B.b WT) but did not emerge where DDNB buffer alone (Fig, 1A, DDNB) or heat killed HI-A mutant were inoculated (Fig, 1A, Heat Killed HI-A). Additionally, the effects of B. bacteriovorus HI mutants on E. coli biofilms were measured. E. coli biofilms (comprised of ~1 × 108 cfu/well) were formed in 96 well microtiter plates for ~18 hrs. Thereafter the medium was removed and the wells were washed with DDNB medium as described in the Materials and Methods. The E. coli biofilms were exposed for 48 hr to the HI mutants, B. bacteriovorus lysate or DDNB. As shown in Fig. 1B (pre-treatment), the untreated 18 hr-old biofilm was easily visualized with CV-staining. Treatment with 1 × 107 pfu of B. bacteriovorus (Fig 1B, B.b WT) or 1 × 107 cfu HI mutants (Fig. 1B, HI-A, B, C) markedly reduced the CV-staining compared to the DDNB or heat killed HI-A control (Fig. 1B, DDNB, and Heat Killed HI-A). Quantification of the effect of B. bacteriovorus on E. coli biofilms over time revealed a 69% reduction in CV staining at 24 hr post-treatment and an 81% reduction after 48 hr (Fig. 1C, B.b WT), compared to the initial time point (pre-treatment). A reduction of 63%, 55%, and 52% was observed following a 24 hr exposure period to HI mutants A, B, C, and a decrease of 70%, 62%, and 63% following 48 hr of incubation (Fig. 1C, HI-A, B, C). In contrast, only a 22% and 16.4% reduction in CV staining was measured after 48 hr in the control sample treated with DDNB and heat killed HI-A respectively (Fig. 1C, DDNB, and Heat Killed HI-A). The ability of the HI mutants to reduce host cells grown planktonicly was also examined in standard induced lysates. All HI mutants, as well as B. bacteriovorus were able to reduce the planktonic population by ~5 logs in the first 24 hr of predation with no reduction occurring when DDNB alone or HI heat killed mutant A was added (Fig. 1D).

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