Abstract
Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a source of nutrients, finally dividing into individual cells (Sockett, 2009). Due to its obligatory predatory lifestyle, manipulation of Bdellovibrio requires two-member culturing techniques using selected prey microorganisms (Lambert et al., 2003). In this protocol, we describe a detailed workflow to grow and quantify B. bacteriovorus HD100 and its predatory ability, to easily carry out these laborious and time-consuming techniques.
Keywords: Bdellovibrio bacteriovorus, Predatory bacteria, Predatory quantification
Background
In the last years, Bdellovibrio has attracted the interest of the scientific community and several applications have been developed, such as evolution studies (Davidov and Jurkevitch, 2009), identification of new biocatalysts (Martínez et al., 2012), therapeutic applications (Atterbury et al., 2011), or biotechnological applications using Bdellovibrio as a lytic agent for the recovery of value added intracellular bioproducts (Martínez et al., 2016). Due to the growing interest in Bdellovibrio, different indirect methods to quantify this predatory bacterium have been developed (Mahmoud et al., 2007; Lambert and Sockett, 2008; Van Essche et al., 2009). However, direct quantification of Bdellovibrio via double-layer method is still necessary to thoroughly characterize Bdellovibrio predatory capability. Here, we describe a well-established, reliable, and broadly used method that allows Bdellovibrio cell number quantification in predatory co-cultures.
Materials and Reagents
Equipment
Procedure
Data analysis
A representative graph of the predatory activity of Bdellovibrio is shown below (Figure 2). Predator and prey cell number at the beginning of the experiment (0 h) and after 24 h of predation upon P. putida KT2440 are represented. Figure 2. Predation profile to study the predatory capability of B. bacteriovorus HD100 upon P. putida KT2440. A. Schematic representation of the co-culture involving B. bacteriovorus HD100 and the control culture of P. putida without predator. B. Viability of the prey and predator cells at the beginning of the experiment (0 h) and after 24 h of incubation. The purple bars represent the viability of the prey cells and the orange bars correspond to the predator viable cell number. Error bars indicate the standard deviation of the mean (n ≥ 3).
Notes
Recipes
Acknowledgments
This protocol was modified and adapted from a predatory assay previously described (Martínez et al., 2016; Lambert et al., 2003). This work was funded by the EU Seventh Framework Programme under grant agreement No. 311815 (SYNPOL), and by grants from the Comunidad de Madrid (P2013/MIT2807) and the Spanish Ministerio de Economía y Competitividad, (BIO2010-21049, BIO2013-44878-R).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.