Members of the Corynebacteria-Mycobacteria-Nocardia-Rhodococcus (CMNR) group of bacteria are best recognized for their impact on human health and they include the causative agent of tuberculosis (TB; Mycobacterium tuberculosis), leprosy (Mycobacterium leprae) and diphtheria (Corynebacterium diptheriae). However, many members of this group are also of great significance to agriculture, biotechnology, and environmental issues. CMNR bacteria also include animal (Mycobacterium bovis, Rhodococcus equi, and species of Corynebacteria and Nocardia) and plant (Rhodococcus) pathogens with a broad host range. They also are widely used in industry. Examples include Corynebacterium glutamicum which is used for large-scale manufacturing of amino acids such as L-glutamate and bacteria that are known to accumulate triacyglycerols (TAGs), oils that are the direct building blocks for biodiesel. Furthermore, some CMNR bacteria are also used for a process termed bioremediation due to their ability to break down harmful and persistent pollutants in the environment.
A common theme among the CMNR bacteria is a lipid-rich cell envelope that contains fatty acids and lipids that are distinct and not found in other bacteria. This complexity is reflected in the relatively large proportion of lipid metabolism related genes in the genomes of these bacteria. The diversity of habitats colonized by CMNR bacteria is rooted in their ability to adapt to new environments by dynamically altering the composition of complex fatty acids in their unique cell envelope. This proposal will test the hypothesis that depending on the environment and available substrates, the CMNR bacteria selectively alter the composition of their cell envelope. We propose that the bacteria achieve by using expanded families of genes involved in the uptake and biosynthesis of cell envelope 'building blocks'. To elucidate how CMNR bacteria achieve this we will first test the ability of a model CMNR bacterium, Mycobacterium smegmatis, to grow on different substrates that can potentially act as 'building blocks'. We will select growth conditions that lead to cell envelope alterations and determine the transciptome (a global picture of the levels to which genes are expressed) of M. smegmatis growing under the select conditions. The information obtained from these studies will then be analysed using in-house computational tools (a program called EGRIN 2.0) to generate models of gene regulatory networks. In other words, we will 'reverse engineer' these networks in the model bacterium. This model will subsequently allow us, by comparing published genomes, to characterize common and unique regulatory mechanisms across CMNR bacteria. Model predictions will be tested by analyzing consequences of specific gene deletions on cell envelope composition of representative CMNR bacteria under relevant conditions. This inter-institutional proposal combines the complementary expertise of the US and UK based research groups. The systems biology expertise of the US-based PI with the UK-based PI's expertise in characterizing CMNR bacteria lipid metabolism and cell envelope biochemistry will allow us to address the fundamental and biotechnologically important question of how CMNR bacteria adapt to new environments.

Technical Abstract:
CMNR bacteria adapt to diverse habitats by using paralogous enzymes in different combinations to selectively catabolize different substrates and alter the composition of their cell envelope. Our proof-of-concept studies show that signatures of these varied conditional combinations are discernible at the level of transcriptional regulation. Therefore, we will apply a systems biology approach to delineate how the CMNR bacterium Mycobacterium smegmatis conditionally alters interactions among paralogs of uptake and biosynthesis enzymes to match cell envelope composition to specific environments. We will use the following approaches to address specific objectives:

1) To determine which growth substrates and environmental transitions induce alterations to cell envelope composition, the effects on growth characteristics and cell wall composition of M. smegmatis will be determined by growing the bacterium in 10 substrates, in 5 different environmental conditions.

2) We will measure temporal changes in genome-wide mRNA levels during physiological transitions of M. smegmatis (above studies) to reverse engineer the underlying gene regulatory network that mediates cell envelope changes. Expression data will be mined using established network inference algorithms, to elucidate the organization of genes into conditionally co-regulated modules (corems), and infer the topology of transcriptional and environmental influences that mediate changes in expression and composition of genes within each corem. In subsequent iterations, model-driven characterization of novel strains will generate new data and propel model refinement.

3) Network model will be used to characterize conserved/specialized mechanisms for regulating cell wall biogenesis across CMNR bacteria. Fully sequenced 144 bacterial genomes will be used to develop new methods for mapping/comparing regulatory network topology in an evolutionary context. Hypotheses will be tested using knockout strains of M.smegmatis.

Potential Impact:
1) Direct Impact: The proposed research will have a direct impact on research on CMNR bacteria and their adaptation to dynamic environments. The immediate beneficiaries will be the two PI's research groups in particular, and on a wider scale the research community. The outcomes from our proposed project will have the potential to further our understanding of the biology of CMNR bacteria, thus having an impact on researchers working with these bacteria. Our work will also contribute to the development of Systems Biology-based approaches to studying bacterial adaptation and bacterial transcription. And finally, the computational skills and tool sets used in this study will be beneficial for computational biologists.

2) Training: The PDRAs employed on this project will be joining the Institute of Microbiology and Infection (IMI, Birmingham) and the Institute of Systems Biology (ISB, Seattle), institutions that host internationally competitive expertise in the biology of microbes and the use of systems approaches to biology, giving the PDRAs exposure to world class research. The PDRAs will also gain training in a wide range of methodologies via collaborations and training exchanges between the two groups, with the PDRAs facilitating the contribution of new ideas to this partnership.

3) Building international links: The BBSRCs strategic plan includes the enabling of partnerships including those that 'maximise the UK's interests both in the EU and worldwide by fostering international relations and links with counterpart organisations overseas'. The work outlined in this proposal allows us to start this process at an institutional level, building collaborative links with a leading US-based research institution, with the potential to expand these links with other groups at the two institutions.

4) Socio-economic impact:
(i) Biotechnology Industry: CMNR bacteria are widely used in industry, including Corynebacterium glutamicum which is used for large-scale manufacturing of amino acids such as L-glutamate and L-lysine. Findings from our studies could potentially inform approaches to increasing product yields by choosing appropriate substrates and growth conditions.
(ii) The UK farming and agriculture industry: the CMNR bacterium Mycobacterium bovis, the causative agent of bovine tuberculosis, cost the UK taxpayer nearly £100 million, while costs to farmers are estimated to have run to tens of millions of pounds (Defra). Some CMNR bacteria are also plant pathogens. Thus, any furthering of our understanding of the biology of these pathogens will impact efforts for developing preventive measures.
(iii) Pharmaceutical Industry and Global Health: The causative agents of tuberculosis, diphtheria, leprosy amongst others are all CMNR bacteria and thus the long term impact on global health are broad, and so are the impacts on developing therapies for these diseases
(iv) General public: In the long term, the general public will be the ultimate beneficiaries, both through the exploitation of useful CMNR bacteria and the targeting of pathogenic CMNR bacteria that affect animal and human health. In the short term, they will be able to access the outputs of our research via our outreach activities which are outlined in the 'Pathways to Impact' document.