microgen - Bdellovibrio bacteriovorus genome sequencing project

Fig. 1. Bdellovibrio attached to a cell of Pseudomonas.

The goal of this project is to sequence and annotate the genome of Bdellovibrio bacteriovorus strain W. Bdellovibrio is especially unique among all the differentiating species of bacteria because it undergoes morphogenetic changes as a part of a life-cycle that is dependent upon attacking and invading gram negative bacteria for food (Fig. 1). Every Bdellovibrio isolated to date has been obligatorily dependent (host dependent - HD) upon a host (prey) cell for growth to carry out its life cycle. However, host-independent (HI) strains also exist (Fig 2) that exhibit the same morphogenetic changes during growth in laboratory media as in the E. coli periplasm (attack cell ' filamentous cell ' fragmenting filament/lysis ' attack phase). Growth in the axenic state results in a 4-6 fold increase in number over the host dependent state. Single step revertants have been isolated, but never to a completely obligatorily host dependent state. That is, the revertants are facultative and able to grow in either mode. Strain W is only one of 2 known cyst-forming isolates reported. Bdellocysts do not develop outside of the prey cell, but form only when periplasmic and remain within the prey cell ghost.

The cyst-like cell expands in size to 2-3 times the diameter of the vegetative Bdellovibrio due to the deposition of additional layers of peptidoglycan. Mature bdellocysts (Fig. 3) are fairly resistant to desiccation. In fact, viable Bdellovibrios have been isolated from soils that were air dried and stored in a 2% moisture environment for 2 years. Utilizing this property, the effectiveness and persistence of Bdellovibrio predation as a biological control agent (and biodefense agent) can be enhanced to useful levels.

Fig. 2. Host-dependent and host-independent life cycle of
Bdellovibrio bacteriovorus.

Under the proper conditions, the bdellocysts germinate by breaking down the outer peptidoglycan layer. The outer peptidoglycan layer of the dormant cyst appears to be uniformly broken down eventually liberating a single, fully mature, motile attack-phase vegetative cell from the prey cell ghost.

The genome size for B. bacteriovorus strain W has been measured by pulsed-field gel electrophoresis at 2.1 x 10⁶ base pairs, but with all enzymes tested, there are too many restriction fragments to generate a physical map (Fig. 4). Little else is known about the organization, replication and ability to manipulate the chromosome.

There remains a plethora of additional biological questions to be answered that range from the mechanism of HI growth to those issues surrounding attachment, invasion, growth and lysis of prey cells and to the regulation of cellular metabolic events associated with growth in both conditions.

We eventually hope to understand and link two seemingly unrelated processes, bacterial predation and morphogenesis. Therefore, after sequencing the genome, the ultimate goals of this project are (a) to determine the genes that are transcriptionally activated or down regulated at each stage of development and during HD/HI growth of the organism and (b) to assess their role in the developmental cycle of the organism.

Fig. 3. Life cycle during cyst formation by strain W.

Fig. 4. Restriction digest of 2 strains of B. bacteriovorus.