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Applied and Environmental Microbiology, August 2007, p. 4717-4724, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.00640-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of a Novel Genetic System To Create Markerless Deletion Mutants of Bdellovibrio bacteriovorus{triangledown}

Susan R. Steyert and Silvia A. Pineiro*

Department of Medical and Research Technology, School of Medicine, University of Maryland, Allied Health Building, 100 Penn Street, Baltimore, Maryland 21201

Received 20 March 2007/ Accepted 4 June 2007

Bdellovibrio bacteriovorus is a species of unique obligate predatory bacteria that utilize gram-negative bacteria as prey. Their life cycle alternates between a motile extracellular phase and a growth phase within the prey cell periplasm. The mechanism of prey cell invasion and the genetic networks and regulation during the life cycle have not been elucidated. The obligate predatory nature of the B. bacteriovorus life cycle suggests the use of this bacterium in potential applications involving pathogen control but adds complexity to the development of practical genetic systems that can be used to determine gene function. This work reports the development of a genetic technique for allelic exchange or gene inactivation by construction of in-frame markerless deletion mutants including the use of a counterselectable marker in B. bacteriovorus. A suicide plasmid carrying the sacB gene for counterselection was used to inactivate the strB gene in B. bacteriovorus HD100 by an in-frame deletion. Despite the inactivation of the strB gene, B. bacteriovorus was found to retain resistance to high concentrations of streptomycin. The stability of a plasmid for use in complementation experiments was also investigated, and it was determined that pMMB206 replicates autonomously in B. bacteriovorus. Development of this practical genetic system now facilitates the study of B. bacteriovorus at the molecular level and will aid in understanding the regulatory networks and gene function in this fascinating predatory bacterium.

* Corresponding author. Mailing address: Department of Medical and Research Technology, School of Medicine, University of Maryland, 100 Penn Street, AHB, Office 415A, Lab 450, Baltimore, MD 21201. Phone: (410) 706-3773. Fax: (410) 706-0073. E-mail: spineiro{at}som.umaryland.edu

{triangledown} Published ahead of print on 8 June 2007.

Applied and Environmental Microbiology, August 2007, p. 4717-4724, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.00640-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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