Nonclassical protein secretion by bacteria.

Free-living microbes must be able to sense, make sense of, respond to, and exploit their outside environment. Microscopic, single-celled bacteria often have very nimble and varied responses to the outside environment. Some are opportunistic pathogens, so they can immediately sense when they have come into contact with a host organism and should infect it. Others have evolved ways to utilise even the most recalcitrant food sources that may be present. Chitin is, after cellulose, the second-most abundant natural polymer on Earth. Present in insect and crustacean exoskeletons, as well as in fungi, chitin is a linear chain of N-Acetyl Glucosamine (GlcNAc) and is completely insoluble and incredibly tough and stable.

Some bacteria have figured out how to use chitin is a carbon and nitrogen source for growth. They secrete different chitinase enzymes all the way out of the cell and, working together outside, these chitinases breakdown the chitin into GlcNAc disaccharides. These are then taken up into the cell and converted to glucose, ammonia and acetic acid. For this system to work, the chitinases must be first made inside the cell and then secreted outside. How bacteria do this is the subject of this research proposal. If we can find out how they do it, maybe we can make them do it more. This will enable us to process food waste into biofuels or other chemicals. Some medical scientists also think that secreted chitinases may help pathogens infect their hosts, so figuring out how to stop enzyme secretion happening may also be of value to society.

In work leading to this proposal we have found the genes essential for chitinase secretion in a bacterium called Serratia marcescens. This bacterium is widespread and well studied, mostly because it can be responsible for heathcare-acquired infections. The chitinase secretion system involves a set of proteins that reside in the cell envelope, surrounding the cell, and an enzyme that controls the permeability of the cell wall to allow the chitinases out. This research project will find out how the system works because nobody knows at the moment.

Classically, proteins destined for secretion could be predicted by the presence of cleavable signal peptides in their amino acid sequences. However, there are increasing examples of nonclassical secretion systems that do not strictly follow this dogma. In bacteria at least nine different protein secretion pathways have been described, some closely related to bacteriophage systems. Serratia marcescens secretes at least four different chitinase enzymes. These allow S. marcescens to utilise chitin as sole carbon and nitrogen source, and may also have a role in pathogenicity. Transposon mutagenesis carried out be us identified an operon (chiWXYZ) that is essential for chitinase secretion by S. marcescens. In cells lacking chiW or chiX the chitinases accumulate in the periplasm and do not cross the outer membrane. A regulator, ChiR, was also identified and overproduction of this protein activates chitinase secretion in all cells. The surprising aspect of chiWXYZ is that it is related to a bacteriophage lysis cassette. ChiW is a holin-like inner membrane protein, ChiX is an L-Ala D-Glu endopeptidase, ChiY is an inner membrane 'spanin' and ChiZ is an outer membrane lipoprotein. The physiological role of ChiWXYZ is not cell lysis, but instead protein secretion. This project will address the relationship between ChiX and ChiW, which we hypothesise acts as a gated channel for the endopeptidase. It will define the molecular features of both ChiW and ChiX that govern chitinase secretion. The project will also establish the roles of ChiY and ChiZ on the secretion pathway and address how they work together. In addition, the project will establish the limits of the role of ChiR in S. marcescens physiology. As well as chitinase and chiWXYZ regulation, our data suggest ChiR also controls genes needed for metabolism of nitrogen componds. Taken altogether, this project will generate deep new knowledge of bacterial physiology and metabolism that could be applied or exploited.

Who will benefit, why and how?

The BBSRC STRATEGIC PLAN highlights 'WORLD CLASS BIOSCIENCE' as of paramount importance and this work builds on high-impact research and is at the forefront of the protein secretion field. This project will provide new knowledge that can be bolted on to industrial fermentation processes, helping to maintain the UK's lead in this area of microbial metabolic and biochemical engineering, and also provide know-how that will benefit an emerging 'circular economy'.

The BBSRC 'BIOENERGY AND INDUSTRIAL BIOTECHNOLOGY' Strategic Research Priority 2 is also relevant as this technology has the long-term potential to promote the efficient use of food waste as biomass for biofuel or biochemical production. It is a new first step in being able to utilise chitin as a potential feedstock.

The research is also relevant to efforts in finding new targets for new antibacterials. Many bacterial pathogens secrete effector molecules, which are usually proteins, to escape the host immune system or to modify the biochemistry of the host cell. Bacterial chitinases, and human chitin-like proteins, have an increasingly important, and intriguing, role in the infection process and in inflammation. The bacterial chitinases, and chitin binding proteins, are believed to facilitate the initial attachment of pathogenic bacteria onto host cells.

As well as meeting UK government priorities, this project will also be of interest to a large number, and a diverse cross-section, of academic researchers, industrialists, and students of microbiology and biochemistry. Applied scientists and the biotechnology industry, who are interested in producing degradative enzymes of industrial relevance will benefit.

Those individuals trained as a direct result of their involvement in this diverse research project will benefit enormously at a personal level and the UK science community will benefit as a result of that training in the medium term. Postdoctoral research assistants will have access to training in transferable skills for the purposes of career and personal development. The University of Dundee is committed to such training and mentoring of research staff and employ an Office for Professional Development whose remit is the provision of a range of courses that all postdoctoral researchers are encouraged to attend.

The results and intellectual property from this study will be protected as appropriate before being disseminated at international conferences and in high quality publications. Strains and other resources will be made available as appropriate.

Finally, the general public will benefit. It is important that members of the public are aware and supportive of how tax payers' money is spent on scientific research. Therefore as part of our work on this project, we will engage with local communities, through face-to-face discussion of our work and family-focussed scientific event days. Our 'Magnificent Microbes' event is now a regular feature in Dundee and is held over two consecutive days in partnership with the Dundee Science Centre. This event will run in the spring of 2018. All staff employed on this project will fully participate in this event.