Characterisation of a sigma factor with an on-board iron-responsive regulatory domain

The Burkholderia are a diverse group of bacteria that live in the environment, particularly in the soil and in association with plants. They are important for a number of reasons. First, they have the ability to metabolise a wide variety of chemicals, and this makes them potentially useful in bioremediation and detoxification of contaminated environments. They are also the source of a variety of interesting compounds that have medical or pharmacological importance. Several species are phytopathogenic (cause disease in plants) while others serve to protect plants from a variety of microbial pathogens and thereby increase the growth yield of crop plants. In recent decades they have come to prominence by virtue of the ability of some members of this group to cause serious infections in humans and animals. Such infections are extremely difficult to treat due to the high level of antibiotic resistance exhibited by Burkholderia.

As with the majority of living things, Burkholderia require iron to grow and they possess very efficient mechanisms to obtain iron from the environment and from the human host. Therefore, the iron acquisition systems of Burkholderia may serve as a target for future development of drugs to target these bacteria. They may also serve as a means for controlling the growth of plant pathogenic or plant growth-promoting Burkholderia. To this end, we are investigating the mechanisms of iron uptake and its regulation in this group of bacteria. We have identified a unique mechanism by which some species of Burkholderia sense and respond to iron in the environment. Specifically, a particular regulatory element consisting of a protein, known as a sigma factor, that targets particular sets of genes for expression is uniquely able to respond to iron directly. The purpose of this project is to apply a range of molecular approaches to investigate the mechanism by which this sigma factor is able to sense and respond to iron. The information gained from this study will permit a greater understanding of gene expression and regulation in bacteria and may serve as a springboard for the development of intervention strategies to control the growth of Burkholderia either in the environment or in animal and human hosts.

Bacterial extracytoplasmic function (ECF) sigma factors are usually partnered by a membrane-bound regulatory protein that senses extracellular stimuli and either inhibits or stimulates the activity of its cognate ECF sigma factor. OrbS belongs to the iron-starvation group of ECF sigma factors and is present in many members of the diverse and important genus Burkholderia, where it serves to activate transcription of siderophore biosynthesis and utilisation genes under iron starvation conditions. The activity of OrbS is stimulated under conditions of decreased iron availability, but the mechanism by which post-translational regulation of OrbS occurs is not through the action of a sigma factor regulatory protein. Unusually, OrbS possesses a cysteine-rich domain located C-terminal to the region that is predicted to interact with the upstream promoter element (the -35 region). We suggest that OrbS has an on-board iron sensory domain that controls OrbS-dependent gene expression.

The aim is to elucidate the mechanism by which iron regulates OrbS activity in the absence of a cognate regulatory protein. Specifically, we will demonstrate that iron interacts with OrbS and determine the nature of the resultant OrbS-iron complex, i.e. whether mononuclear iron or an iron-sulfur cluster is involved, and the characteristics of such a complex. We will also elucidate the mechanism by which iron-binding impairs the transcriptional proficiency of OrbS by analysing the effect of iron binding on the stability of OrbS, its assembly into RNA polymerase holoenzyme and OrbS-dependent transcription initiation. Some species of Burkholderia cause significant animal and human suffering or are phytopathogens, whilst others are being assessed as plant-growth promoting agents. In both settings, iron-acquisition by the bacterium is crucial and thus our work has the potential to impact on two key BBSRC research priorities, viz. 'Basic science underpinning health' and 'Food security'.

This project is an investigation into the mechanism of iron-dependent autoregulation of a bacterial transcription factor, the first example of its kind described. It will include an analysis of the assembly and disassembly of the metallocentre in response to iron availability and the elucidation of the mechanism by which iron-binding interferes with promoter utilisation by the transcription factor. The foreseeable beneficiaries in the UK and internationally include:

1. Early-career bio-scientists for the biotechnology, pharmaceutical and public sector laboratories as well as other professions that require individuals possessing an in-depth knowledge of molecular biology and microbiology such as research councils, Health and Safety Executive, investment companies and patent authorities/organisations seeking expertise in the biotechnology industry. They will benefit by high quality training provided in the laboratories of the applicants.

2. The academic community at large will benefit from an improved overall knowledge of bacterial physiology and its regulation, and particularly their understanding of the biology of an extremely important genus of bacteria - Burkholderia. These bacteria are prevalent in the environment and some species offer enormous potential benefits for the national and international community (biocontrol and bioremediation) while others offer a threat (human and plant diseases, bioterrorism). Iron acquisition is an important contributor to their success and so the knowledge gained from this investigation is likely to have diverse beneficial consequences.

3. Research scientists investigating Burkholderia biology and iron acquisition in other bacteria will benefit from the establishment of new knowledge, experimental tools and bacterial strains.

4. The agri-food industry and the general population who may benefit from improved crop yields ('food security') and reduced use of pesticides as a result of the introduction of genetically modified Burkholderia biocontrol strains. Although a long term goal, a knowledge of the mechanism by which OrbS senses and responds to iron will inform strategies devised to improve the ability of plant-beneficial species to more efficiently acquire iron from the rhizosphere and thereby stymie the growth of phyopathogenic microbes occupying the same niche.

5. Health services and patients susceptible to opportunistic infections by Burkholderia will benefit from novel intervention strategies for the treatment of infections by these organisms. Such strategies may also have implications for national security due to the potential use of one or two members of this genus as agents of bioterrorism. As iron acquisition is essential for establishment of infections by Burkholderia, increasing our knowledge of iron acquisition mechanisms and their regulation may lead to targeted 'antivirulence' therapies focused on the iron uptake system or its regulatory switch.

6. Local water authorities and municipal councils stand to benefit from the introduction of modified Burkholderia biosensor strains that can detect specific contaminating heavy metal ions in the environment. The ability of thiols to interact with a large variety of transition metals offers the potential for configuring the iron-binding region of OrbS to preferentially bind an alternative metal such as zinc, molybdenum, copper, selenium etc, but more usefully to bind a toxic metal such as arsenic, cadmium or mercury. For example, by linking the modified orbS gene to an OrbS-dependent promoter driving expression of a repressor that is, in turn, coupled with a reporter gene placed under control of the repressor, this system can be used to assess the presence of toxic metal ions.

7. The general public will benefit from better information regarding the importance of fundamental science in advancing quality of life, particularly regarding scientific concepts and issues over which society has an influence.