The role of the dynamin membrane-remodelling proteins in developmentally controlled cell division in Streptomyces

Streptomyces are harmless soil bacteria that resemble moulds in that they are filamentous, rather than unicellular organisms. They are vital to human welfare because they are the source of the vast majority of antibiotics used by doctors to cure infectious diseases, as well as providing us with numerous other medicines used, for example, to treat cancer, and to help organ transplant patients. Streptomyces reproduce themselves by dividing specialised reproductive filaments called 'aerial hyphae' into long chains of spores, in a process called 'morphological differentiation'. Antibiotic production is tightly coordinated with morphological differentiation. Morphological differentiation to form dormant spores requires each aerial hypha to synchronously lay down 50 or more regularly spaced, specialized walls called 'sporulation septa'. These sporulation septa grow in from the sides of the aerial hyphae, with the membrane of the hyphae invaginating around the ingrowing wall, until both membrane and wall fuse in the middle. We have discovered two proteins called 'dynamins' that are required for this massive cell division event to take place normally. The dynamin proteins are involved in remodeling the membranes as they invaginate and fuse, but somehow they are also required to stabilize the molecular machinery that synthesizes the ingrowing cell wall. The aim of this grant application is to discover how the dynamins achieve these functions at the molecular level. We also know that the dynamins are present in the closely related (but experimentally very difficult) bacterium Mycobacterium tuberculosis, which causes the fatal disease tuberculosis, and that the cell division machinery we will be studying in Streptomyces is closely related to the cell division machinery in this killer bug. Thus, this work may also provide significant insights into the cell division machinery of another bacterium important to human health. Finding ways to inhibit cell division in pathogenic bacteria could lead to more effective treatments for the diseases they cause.

The objectives will be addressed through a multidisciplinary programme incorporating cell biology, genetics, biochemistry and structural biology.

OBJECTIVE 1. IDENTIFYING PROTEINS THAT INTERACT WITH DynA and DynB.
We have already identified two Dynamin-Interacting Partners (DIPs), Dip1 and Dip2, by screening targets of WhiAB, the two regulators that co-control initiation of sporulation septation. Further DIPs will be identified by:
(i) Shotgun bacterial 2-hybrid (B2H) screening.
(ii) Targeted B2H screens against SepF1, SepF2, SepF3, SsgA and SsgB.
(iii) Co-IP assays using 3xFLAG-tagged dynA and dynB alleles.
We will also:
(iv) Use targeted yeast 2H screens against DynA, DynB, Dip1, Dip2, SepF1, SepF2, SepF3, SsgA and SsgB to identify FtsZ-interacting proteins (FtsZ is not functional in the B2H system).

OBJECTIVE 2. DISSECTING DYNAMIN-PARTNER INTERACTIONS AND THEIR ROLE IN CELL DIVISION.
Having identified DIPs, we will conduct experiments to understand how they contribute to developmentally controlled cell division through:
(i) Construction and phenotypic characterisation of DIP mutants.
(ii) Localisation of DIPs in sporulation movies.
(iii) Analysing interactions between the dynamins, DIPs, FtsZ and other divisome components by introducing fluorescent fusions into mutants to determine how loss of the encoded protein affects the localisation and dynamics of the fusions.

OBJECTIVE 3. ANALYSING THE INTERACTION OF THE DYNAMINS WITH LIPID BILAYERS.
We will assay DynA, DynB, and DynA+DynB for:

(i) GTP binding.
(ii) GTPase activity; stimulation of GTP hydrolysis by liposomes.
(iii) GTP-dependent membrane tethering.
(iv) Ordered self-assembly on liposomes (visualized by EM).
(v) The ability to fuse membranes.

OBJECTIVE 4. SOLVING THE STRUCTURE OF DynA AND DynB IN COMPLEX. Dr Low (Imperial College) will use a combination of X-ray crystallography and cryo-electron microscopy to generate atomic structures of the DynA-DynB complex.

WHO WILL BENEFIT FROM THIS RESEARCH? The outputs of this research will be of value to fundamental scientists, to the pharmaceutical industry, and ultimately to the health sector and thus to patients.

HOW WILL THEY BENEFIT FROM THIS RESEARCH? Streptomycetes account for ~80% of commercially important antibiotics used in human medicine, and are also a rich source of other types of bioactive molecules such as anticancer agents and immunosuppressants, currently accounting for ~$40 billion of revenue annually in the pharmaceutical industry worldwide. Importantly, the production of antibiotics is not constitutive, Rather, this 'physiological differentiation' is tightly coordinated, both temporally and genetically, with morphological differentiation into dormant spores. There is no doubt, therefore, that the full exploitation of Streptomyces for the production and discovery of antibiotics will depend on a much better understanding of the developmental biology of the organism as a whole. Thus, the proposed fundamental study on developmentally controlled cell division in Streptomyces will be of direct interest to companies manufacturing streptomycete antibiotics. Further, many of the components of Streptomyces divisome under study here are also components of the divisome in medically important actinomycete relatives like the pathogens M. tuberculosis and C. diphtheriae. A unique advantage of working on these proteins in Streptomyces is that cell division is non-essential in filamentous bacteria like Streptomyces so that, for example, ftsZ null mutants are viable, whereas equivalent mutations are lethal in unicellular bacteria like M. tuberculosis and C. diphtheriae. New drugs that target the actinomycete divisome could be effective treatments for M. tuberculosis and C. diphtheriae infections. Thus the proposed work has potential commercial and medical relevance through multiple paths.

WHAT WILL BE DONE TO ENSURE THAT THEY BENEFIT FROM THIS RESEARCH? Outputs with potential commercial impact will be identified during regular reviews of progress. Discoveries with potential commercial implications will be discussed (with a view to patenting) with Plant Biosciences Ltd (PBL), a technology transfer company jointly owned by the Gatsby Foundation and the JIC. The purpose of PBL is to bring the results of research in plant and microbial sciences at the Centre into public use for public benefit through commercial exploitation. PBL meets all patent filing, marketing and licensing expenses in respect of technologies it develops for JIC. Streptomyces research is prominent in PBL's portfolio. As an illustration, two spin-out companies have been established based on JIC Streptomyces group patents: Novacta Biosystems Ltd (founded 2003), and Procarta Biosystems (founded 2008). Thus, there are established routes for delivery of IP arising from Streptomyces research at the JIC.

Inspired by the professional video filmed as part of our recent publication in the Journal of Visualized Experiments, we will commission a professionally-made general video to illustrate and advertise the work on Streptomyces bacteria at the John Innes Centre for a general audience (public and schools), and use a digital marketing campaign to draw it to the attention of appropriate audiences.

Mark Buttner and Susan Schlimpert will participate in the JIC Teacher-Scientist Network (TSN), give public lectures on e.g. antibiotic resistance and the need for new antibiotics, make presentations to the Friends of the JIC, and to the general public through open days.

JIC has an excellent External Relations Department (http://www.jic.ac.uk/corporate/media-and-public/index.htm) and, where appropriate, we will work proactively with them to approach and interact with the press and broadcast media to publicise this scientific area in general and the outputs of the grant.