Genetics of the switch from predatory to axenic growth in the living antibiotic Bdellovibrio bacteriovorus

Bdellovibrio are small, human-friendly, predatory bacteria which invade the cells of other pathogenic bacteria (such as ones that give old people infections in pressure sores and diabetic ulcers, and bacteria that infect crop plants) and kill them. Bdellovibrio have no activities against human, animal or plant cells but they are naturally good at killing other bacteria. Pathogenic bacteria do not have simple cell surface receptors for Bdellovibrio attachment and invasion and so it is not easy for them to develop resistance to the Bdellovibrio (unlike the situation with conventional antibiotics) Bdellovibrio were discovered in the 1960s and recently we and others have been researching their genes and genomes and trying to understand how they may be useful to humans. We propose that they can be used in a cream or a spray to apply to infected wounds in humans and also to be sprayed on infected crop plants to kill off infections crop rot bacteria. Bdellovibrio are almost unique in being predatory bacteria, most bacteria grow on foods and do not invade live bacteria. Bdellovibrio actually have a large 3.8Mb genome (which we helped to sequence in 2004) and have got both the genes to grow like normal bacteria, and the genes to be predatory.To be efficient at killing pathogens Bdellovibrio will have to lose the ability to grow normally on food like regular bacteria, because that ability allows them to grow in serum in wounds and in sap and on soil around plants, without needing to be predatory and kill the target pathogenic bacteria that are causing the infection. So, the point of our project is to work out how they grow non-predatorily (as so-called HI strains), we will work out which genes code for these properties and try to inactivate them, without impairing predatory growth (which we want to use to kill the pathogens). Previous scientists have published one nice paper in 1992 showing that one gene, in a region of the genome called the hit locus, has changes within it when the Bdellovibrio are growing non predatorily as HIs and that this might be partly responsible, but another paper in 2001 showed that not all Bdellovibrio that were growing non-predatorily had changes in this locus, so there are more important genes to find in the process too. No-one has yet found out what the hit locus gene does and how it might control HI growth- we want to do this here and to see how genetically stable HI strains may be if we produce as living antibiotics. We have carried out previous research that has shown all the genes switched on from the genome when the Bdellovibrio are growing predatorily and when they are growing as HIs; there are lots of gene differences and some are specifically switched on just to grow as HIs. We want to investigate what these genes do, especially some that regulate the switching on of groups of genes- we may be able to delete one of these regulator genes and switch off the whole HI growing abilities, leaving an obligately predatory Bdellovibrio for use as a living antibiotic. We have also already been able to isolate a mutant Bdellovibrio by chance, that we found to be unable to grow as an HI and which is therefore obligately predatory. This strain has a defect in obtaining iron for growth from complex molecules, yet can grow fine in the predatory growth mode, probably because it is able to free the iron from prey bacteria by digesting their proteins. Thus this strain shows that we will be able to knock out genes in Bdellovibrio that control HI growth without abolishing the ability to grow predatorily. In our project we want to profile the genes that are expressed in our iron-scavenging mutant that can't grow as an HI, to try to understand what is wrong with it. The long term idea is to develop Bdellovibrio as a real new treatment for human, animal and plant disease and this is one of the important first steps

Bdellovibrio are Gram-negative bacteria which invade and kill other Gram-negative bacteria, including many pathogens of man, animals and plants, and as such have great potential for use as 'living antibiotics'. Unlike phage, they do not attach to their prey via a single surface receptor and so resistance is not easily developed. We envisage the use of Bdellovibrio as a topical treatment for Gram-negative infections of burn wounds and pressure sores. Bdellovibrio have a large genome for a predator of 3.8Mbp and appear to have adapted from a saprophytic lifestyle still retaining the capacity to grow axenically host independently (HI) Obligate predators incapable of axenic growth should be more efficient for antimicrobial action and would remove any concerns of persistence of the applied Bdellovibrio after successful application. The aim here is to identify the control of HI growth as distinct from predatory growth and to disrupt the former without disrupting the latter. In 1992 Thomashow identified the hit locus as being mutated in their HI strains Barel et al in 2001 showed that while this was often the case, it was not in all HI strains. Here we hope to determine the role of the hit locus by examining the cellular location of its product, its interactions with other proteins and by identifying mutations in other genes in HI strains. We have transcription analyses in both predatory and HI Bdellovibrio and from this identified regulators likely involved in the HI phenotype. By analysing their regulons and the phenotype of mutations in these genes we hope to gain insight into the regulation of the HI phenotype and create a stable obligately predatory mutant strain. We have already identified one such strain, deficient in siderophore production which is obligately predatory and cannot grow in serum, and will test its phenotype futher. The ultimate aim is to develop Bdellovibrio as therapuetics and understanding the nature of the HI phenotype is an important first step