DivIVA Controls Progeny Morphology and Diverse ParA Proteins Regulate Cell Division or Gliding Motility in Bdellovibrio bacteriovorus.

The predatory bacterium B. bacteriovorus grows and divides inside the periplasm of Gram-negative bacteria, forming a structure known as a bdelloplast. Cell division of predators inside the dead prey cell is not by binary fission but instead by synchronous division of a single elongated filamentous cell into odd or even numbers of progeny cells. Bdellovibrio replication and cell division processes are dependent on the finite level of nutrients available from inside the prey bacterium. The filamentous growth and division process of the predator maximizes the number of progeny produced by the finite nutrients in a way that binary fission could not. To learn more about such an unusual growth profile, we studied the role of DivIVA in the growing Bdellovibrio cell. This protein is well known for its link to polar cell growth and spore formation in Gram-positive bacteria, but little is known about its function in a predatory growth context. We show that DivIVA is expressed in the growing B. bacteriovorus cell and controls cell morphology during filamentous cell division, but not the number of progeny produced. Bacterial Two Hybrid (BTH) analysis shows DivIVA may interact with proteins that respond to metabolic indicators of amino-acid biosynthesis or changes in redox state. Such changes may be relevant signals to the predator, indicating the consumption of prey nutrients within the sealed bdelloplast environment. ParA, a chromosome segregation protein, also contributes to bacterial septation in many species. The B. bacteriovorus genome contains three ParA homologs; we identify a canonical ParAB pair required for predatory cell division and show a BTH interaction between a gene product encoded from the same operon as DivIVA with the canonical ParA. The remaining ParA proteins are both expressed in Bdellovibrio but are not required for predator cell division. Instead, one of these ParA proteins coordinates gliding motility, changing the frequency at which the cells reverse direction. Our work will prime further studies into how one bacterium can co-ordinate its cell division with the destruction of another bacterium that it dwells within.

A novel method to determine antibiotic sensitivity in Bdellovibrio bacteriovorus reveals a DHFR-dependent natural trimethoprim resistance.

Bdellovibrio bacteriovorus is a small Gram-negative bacterium and an obligate predator of other Gram-negative bacteria. Prey resistance to B. bacteriovorus attack is rare and transient. This consideration together with its safety and low immunogenicity makes B. bacteriovorus a valid alternative to antibiotics, especially in the treatment of multidrug resistant pathogens. In this study we developed a novel technique to estimate B. bacteriovorus sensitivity against antibiotics in order to make feasible the development and testing of co-therapies with antibiotics that would increase its antimicrobial efficacy and at the same time reduce the development of drug resistance. Results from tests performed with this technique show that among all tested antibiotics, trimethoprim has the lowest antimicrobial effect on B. bacteriovorus. Additional experiments revealed that the mechanism of trimethoprim resistance in B. bacteriovorus depends on the low affinity of this compound for the B. bacteriovorus dihydrofolate reductase (Bd DHFR).

Activity of Bdellovibrio hit locus proteins, Bd0108 and Bd0109, links Type IVa pilus extrusion/retraction status to prey-independent growth signalling.

Bdellovibrio bacteriovorus are facultatively predatory bacteria that grow within gram-negative prey, using pili to invade their periplasmic niche. They also grow prey-independently on organic nutrients after undergoing a reversible switch. The nature of the growth switching mechanism has been elusive, but several independent reports suggested mutations in the hit (host-interaction) locus on the Bdellovibrio genome were associated with the transition to prey-independent growth. Pili are essential for prey entry by Bdellovibrio and sequence analysis of the hit locus predicted that it was part of a cluster of Type IVb pilus-associated genes, containing bd0108 and bd0109. In this study we have deleted the whole bd0108 gene, which is unique to Bdellovibrio, and compared its phenotype to strains containing spontaneous mutations in bd0108 and the common natural 42 bp deletion variant of bd0108. We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type. The pili isolated from these strains were comprised of the Type IVa pilin protein; PilA. Attempts to similarly delete gene bd0109, which like bd0108 encodes a periplasmic/secreted protein, were not successful, suggesting that it is likely to be essential for Bdellovibrio viability in any growth mode. Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108. These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell. We further identify a novel small RNA encoded by the hit locus, the transcription of which is altered in different bd0108 mutation backgrounds.

A transcriptional "Scream" early response of E. coli prey to predatory invasion by Bdellovibrio.

We have transcriptionally profiled the genes differentially expressed in E. coli prey cells when predatorily attacked by Bdellovibrio bacteriovorus just prior to prey cell killing. This is a brief, approximately 20-25 min period when the prey cell is still alive but contains a Bdellovibrio cell in its periplasm or attached to and penetrating its outer membrane. Total RNA was harvested and labelled 15 min after initiating a semi-synchronous infection with an excess of Bdellovibrio preying upon E. coli and hybridised to a macroarray spotted with all predicted ORFs of E. coli. SAM analysis and t-tests were performed on the resulting data and 126 E. coli genes were found to be significantly differentially regulated by the prey upon attack by Bdellovibrio. The results were confirmed by QRT-PCR. Amongst the prey genes upregulated were a variety of general stress response genes, potentially "selfish" genes within or near prophages and transposable elements, and genes responding to damage in the periplasm and osmotic stress. Essentially, the presence of the invading Bdellovibrio and the resulting damage to the prey cell elicited a small "transcriptional scream", but seemingly no specific defensive mechanism with which to counter the Bdellovibrio attack. This supports other studies which do not find Bdellovibrio resistance responses in prey, and bodes well for its use as a "living antibiotic".

Manipulating each MreB of Bdellovibrio bacteriovorus gives diverse morphological and predatory phenotypes.

We studied the two mreB genes, encoding actinlike cytoskeletal elements, in the predatory bacterium Bdellovibrio bacteriovorus. This bacterium enters and replicates within other Gram-negative bacteria by attack-phase Bdellovibrio squeezing through prey outer membrane, residing and growing filamentously in the prey periplasm forming an infective "bdelloplast," and septating after 4 h, once the prey contents are consumed. This lifestyle brings challenges to the Bdellovibrio cytoskeleton. Both mreB genes were essential for viable predatory growth, but C-terminal green fluorescent protein tagging each separately with monomeric teal-fluorescent protein (mTFP) gave two strains with phenotypic changes at different stages in predatory growth and development. MreB1-mTFP cells arrested growth early in bdelloplast formation, despite successful degradation of prey nucleoid. A large population of stalled bdelloplasts formed in predatory cultures and predation proceeded very slowly. A small proportion of bdelloplasts lysed after several days, liberating MreB1-mTFP attack-phase cells of wild-type morphology; this process was aided by subinhibitory concentrations of an MreB-specific inhibitor, A22. MreB2-mTFP, in contrast, was predatory at an almost wild-type rate but yielded attack-phase cells with diverse morphologies, including spherical, elongated, and branched, the first time such phenotypes have been described. Wild-type predatory rates were seen for all but spherical morphotypes, and septation of elongated morphotypes was achieved by the addition of A22.

Predatory lifestyle of Bdellovibrio bacteriovorus.

Bdellovibrio species are naturally predatory, small, motile, Deltaproteobacteria that invade the periplasm of other larger gram-negative bacteria, killing and digesting them. Bdellovibrio grows and divides inside the prey cell, in a structure called a bdelloplast, which then lyses, releasing the Bdellovibrio to prey upon more bacteria. This capability makes Bdellovibrio a potential therapeutic agent, but since its discovery in the 1960s it has not been applied in this way. This review considers what is known postgenomically about Bdellovibrio and its predatory lifestyle, drawing also from what was learned by the excellent microbial physiology work of the early Bdellovibrio researchers. Recent work on the diversity and evolution of predatory bdellovibrios, the role of surface structures in predation, and the ongoing questions about how Bdellovibrio switches between axenic and predatory growth and how its predatory activities may be tempered in the wild, as well as suggestions for future research priorities, are discussed.