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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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Screening for HI transposon mutants defective in biofilm predation. HI transposon mutants were grown in a 96 well microtiter dish. Aliquots were transferred onto a lawn of thickly spread prey cells (plaque predation assays) using a 48-prong multi-well transfer device. The plates were then incubated at 30°C and examined for the formation of a zone of clearing where the mutants were spotted. The arrow indicates the location of a mutant impaired in its ability to form a lytic halo.
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Figure 2: Screening for HI transposon mutants defective in biofilm predation. HI transposon mutants were grown in a 96 well microtiter dish. Aliquots were transferred onto a lawn of thickly spread prey cells (plaque predation assays) using a 48-prong multi-well transfer device. The plates were then incubated at 30°C and examined for the formation of a zone of clearing where the mutants were spotted. The arrow indicates the location of a mutant impaired in its ability to form a lytic halo.

Mentions: To isolate HI mutants defective in biofilm predation, a mariner-based transposon was used to mutagenize B. bacteriovorus HI. Mutant HI colonies were placed into 50 flat-bottom 96 well dishes. For isolating HI mutants impaired in their ability to reduce surface attached bacteria, the HI transposon mutant library was grown in PYE medium for 72 hr. Thereafter, a 96-prong multi-well transfer device was used to transfer aliquots of mutant libraries into a preformed E. coli biofilm (biofilm predation assays) that was developed in 96 well plates or on lawns of prey cells (Fig 2, plaque predation assay). Using this approach 47 HI transposon mutants that were unable to reduce the preformed biofilms (biofilm predation assays) were isolated. These mutants were termed Biofilm Predation Mutants (BPM). No difference in growth rate was observed between BPM mutants and the HI recipient when grown in PYE medium (data not shown).


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)

Screening for HI transposon mutants defective in biofilm predation. HI transposon mutants were grown in a 96 well microtiter dish. Aliquots were transferred onto a lawn of thickly spread prey cells (plaque predation assays) using a 48-prong multi-well transfer device. The plates were then incubated at 30°C and examined for the formation of a zone of clearing where the mutants were spotted. The arrow indicates the location of a mutant impaired in its ability to form a lytic halo.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2277423&req=5

Figure 2: Screening for HI transposon mutants defective in biofilm predation. HI transposon mutants were grown in a 96 well microtiter dish. Aliquots were transferred onto a lawn of thickly spread prey cells (plaque predation assays) using a 48-prong multi-well transfer device. The plates were then incubated at 30°C and examined for the formation of a zone of clearing where the mutants were spotted. The arrow indicates the location of a mutant impaired in its ability to form a lytic halo.
Mentions: To isolate HI mutants defective in biofilm predation, a mariner-based transposon was used to mutagenize B. bacteriovorus HI. Mutant HI colonies were placed into 50 flat-bottom 96 well dishes. For isolating HI mutants impaired in their ability to reduce surface attached bacteria, the HI transposon mutant library was grown in PYE medium for 72 hr. Thereafter, a 96-prong multi-well transfer device was used to transfer aliquots of mutant libraries into a preformed E. coli biofilm (biofilm predation assays) that was developed in 96 well plates or on lawns of prey cells (Fig 2, plaque predation assay). Using this approach 47 HI transposon mutants that were unable to reduce the preformed biofilms (biofilm predation assays) were isolated. These mutants were termed Biofilm Predation Mutants (BPM). No difference in growth rate was observed between BPM mutants and the HI recipient when grown in PYE medium (data not shown).

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